bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular analysis of PHYA in wild‐type and phytochrome A‐deficient mutants of tomato
Authors: Lazarova Galina I. , Kerckhoffs L. Huub J. , Brandstädter Jörg , Matsui Minami , Kendrick Richard E. , Cordonnier‐Pratt Marie‐Michèle , Pratt Lee H. 
Published: March 2003

SummaryTomato (Lycopersicon esculentum Mill., recently redesignated Solanum lycopersicum L.), an agronomically important crop plant, has been adopted as a model species complementary to Arabidopsis in which to characterize the phytochrome family. Here we describe the cloning and molecular characterization of the gene encoding the apoprotein of phytochrome A in wild‐type tomato and in the far‐red‐light‐insensitive (fri1 and fri2) tomato mutants. The physical organization of this gene is similar to that of other angiosperm phytochromes with the four exons of the coding region interrupted by three introns. The pool of transcripts is heterogeneous due to multiple transcription start sites and to three modes of alternative splicing of the 5′ leader. The leader in each alternative transcript carries multiple upstream open reading frames of considerable length. At the genomic level, both fri mutants share an identical base substitution which changes a consensus AG/ to TG/ at the 3′ end of the intron between exons 1 and 2. This mutation leads to aberrant processing of the resultant pre‐mRNA. While most mature transcripts retain the mutated intron, both cryptic splicing and exon skipping were also detected. Cryptic splicing occurred both upstream and downstream from the wild‐type splice site. These observations are consistent with the hypothesis that exon definition in splicing of plant pre‐mRNAs plays a secondary role to that of intron definition. Analysis of the frequency with which potentially functional phytochrome A apoproteins might be produced indicates that both fri1 and fri2 have less than 1% of the wild‐type phytochrome A level.
Stable albinism induced without mutagenesis: a model for ribosome‐free plastid inheritance
Authors: Zubko Mikhajlo K. , Day Anil 
Published: March 2003

SummaryMaternally inherited chlorophyll deficiency, or albinism, is a standard marker in plant cytoplasmic genetics. Its stability is consistent with mutations in the plastid genome. Nuclear mutations inducing plastid ribosome deficiency (PRD) also lead to maternally inherited chlorophyll deficiency. Here we report that stable chlorophyll deficiency can be efficiently generated in cruciferous plants without mutagenesis by a short exposure to spectinomycin, an inhibitor of plastid protein synthesis. We show that the chlorophyll‐deficient phenotype results from a deficiency in plastid ribosomes and plastid translation products. Loss of plastid ribosomes is irreversible. The data suggest that mutations are not essential for generating inheritable PRD. It allows the formulation of a more general model in which stable PRD can be induced by a variety of factors that prevent the formation of functional plastid ribosomes. A non‐mutational mechanism for generating inheritable chlorophyll deficiency has implications for the origin and inheritance of green–white variegation in nature.
Presence of miniature inverted‐repeat transposable elements (MITEs) in the genome ofArabidopsis thaliana: characterisation of theEmigrantfamily of elements
Authors: Casacuberta Elena , Puigdomènech Josep M. Casacuberta Pere , Monfort Amparo 
Published: March 2003

Summary Although the genome of

Arabidopsis thaliana

has a small amount of repetitive DNA, it contains representatives of most classes of mobile elements. However, to date, no miniature inverted‐repeat transposable element (MITE) has been described in this plant. Here, we describe a new family of repeated sequences that we have named

Emigrant

, which are dispersed in the genome of

Arabidopsi

s and fulfil all the requirements of MITEs. These sequences are short, AT‐rich, have terminal inverted repeats (TIRs), and do not seem to have any coding capacity. Evidence for the mobility of

Emigrant

elements has been obtained from the absence of one of these elements in a specific

Arabidopsis

ecotype.

Emigrant

is also present in the genome of different

Brassicae

and its TIRs are 74% identical to those of

Wujin

elements, a recently described family of MITEs from the yellow fever mosquito

Aedes aegypti.
The bifactorial endosperm box of γ‐zein gene: characterisation and function of the Pb3 and GZMcis‐acting elements
Authors: Marzábal Pau , Busk Peter K. , Ludevid M. Dolors , Torrent Margarita 
Published: March 2003

Summary The proximal region of the γ‐zein promoter (γ

Z

) has a functional bifactorial prolamin box element containing two

cis

‐acting elements, a prolamin‐box motif (Pb3) and a GCN4‐like motif (GZM). By particle bombardment of maize endosperms with 5′ deletions and internal deletions of γ

Z

fused to the GUS gene, we have shown that a 135 bp region containing the bifactorial element is involved in the transcriptional activation of the γ

Z

promoter. However, the 135 bp region was unable to activate the γ

Z

promoter in the absence of a 84 bp downstream sequence. Using

in vivo

footprinting and gel mobility shift assays with 15 DAP endosperm nuclear extracts, we have demonstrated the presence of

trans

‐acting factors that interact with Pb3 and GZM target sites. Base‐substitution mutations within Pb3 and GZM decreased transcription activity of the γ

Z

promoter suggesting a co‐ordinated function between the two

cis

‐acting elements. Two additional

cis

‐motifs upstream of the bifactorial prolamin element have been identified: a motif with high homology to the AACA elements of rice glutelin genes and an AZM motif containing an ACGT core which binds nuclear proteins other than the Opaque 2 (O2).
The plant PTS1 receptor: similarities and differences to its human and yeast counterparts
Authors: Wimmer Christine , Schmid Markus , Veenhuis Marten , Gietl Christine 
Published: March 2003

Summary Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon (

Citrullus vulgaris

) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W‐X2‐(E/S)‐(Y/F/Q) in its N‐terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the

Hansenula polymorpha

peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the

Hansenula polymorpha

integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.
The blue light receptor cryptochrome 1 can act independently of phytochrome A and B inArabidopsis thaliana
Authors: Poppe Christoph , Sweere Uta , Drumm‐Herrel Helge , Schäfer Eberhard 
Published: March 2003

Summary Blue light responses in higher plants can be mediated not only by specific blue light receptors, but also by the red/far‐red photoreversible phytochrome system. The question of interdependence between these photoreceptors has been debated over many years. The availability of

Arabidopsis

mutants for the blue light receptor CRY1 and for the two major phytochromes phyA and phyB allows a reinvestigation of this question. The analysis of photocontrol of seed germination, inhibition of hypocotyl growth and anthocyanin accumulation clearly demonstrates that (i) phyA shows a strong control in blue light responses especially at low fluence rates; (ii) phyB mediated induction reactions can be reversed by subsequent blue light irradiations; and (iii) CRY1 mediates blue light controlled inhibition of hypocotyl growth only at fluence rates higher than 5 μmol m–2s–1 and independently of phytochrome A and B.
The maizelilliputianmutation is responsible for disrupted morphogenesis and minute stature
Authors: Dolfini Silvana , Landoni Michela , Consonni Gabriella , Rascio Nicoletta , Vecchia Francesca Dalla , Gavazzi Giuseppe 
Published: March 2003

Summary A recessive maize mutant disrupted in seedling development was isolated following transpositional mutagenesis with

Mutator

. This mutant,

lilliputian
(lil1), exhibits a phenotype consisting of a large primary root, extremely reduced stature, crinkly leaves and has been mapped by TB‐A translocations on the short arm of chromosome 2. Comparative histological analyses revealed an increase in cell number in the mutant root and leaves, as well as an irregularity in cell shapes, suggesting that the altered morphology oflil1roots and leaves is related to an uncoordinated increase in cell divisions and to a defect in division plane alignment. On the other hand, the reduced stature of the mutant plant may be ascribed to a defect in cell elongation.In situhybridization of primary root tissues reveals higher levels of α‐tubulin transcripts in the pericycle and in cells surrounding vascular elements in mutant rather than in wild‐type tissues. These features suggest a role of theLil1gene in the correct functioning of microtubules. Moreover, the fact that the mutation is recognizable even in the dormant embryo and that mutant seedlings are obtained from immature (12 DAP)lil1embryos indicates that the defect in cell division pattern accounting for the disrupted seedling morphology manifests itself even during embryo development.
Sequences within both the N‐ and C‐terminal domains of phytochrome A are required for PFR ubiquitination and degradation
Authors: Clough Richard C. , Jordan‐Beebe Emily T. , Lohman Karin N. , Marita Jane M. , Walker Joseph M. , Gatz Christiane , Vierstra Richard D. 
Published: March 2003

Summary Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr form initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant

PHY

genes

in vitro

and analyzed the Pfr‐specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C‐terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C‐terminus is essential for the form‐dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato

PHYA

and

PHYB

, we found that the N‐terminal half of phyA is also required for Pfr‐specific breakdown. Only those chimeras containing the N‐terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C‐terminal domain is essential for phyA degradation, the N‐terminal domain is responsible for the selective recognition and ubiquitination of Pfr.
Chloroplast Cpn20 forms a tetrameric structure inArabidopsis thaliana
Authors: Koumoto Yasuko , Shimada Tomoo , Kondo Maki , Takao Toshifumi , Shimonishi Yasutsugu , Hara‐Nishimura Ikuko , Nishimura Mikio 
Published: March 2003

Summary Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the
Escherichia coli
GroES, which is a critical regulator of chaperonin‐mediated protein folding. The cDNA for a Cpn20 homologue of
Arabidopsis thaliana
was isolated. It was 958 bp long, encoding a protein of 253 amino acids. The protein was composed of an N‐terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other. The isolated cDNA was constitutively expressed in transgenic tobacco. Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco. A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine‐tagged construct lacking the transit peptide was expressed in
E. coli
and purified by affinity chromatography. Gel‐filtration and cross‐linking analyses showed that the expressed products formed a tetramer. The expressed products could substitute for GroES to assist the refolding of citrate synthase under non‐permissive conditions. The analysis on the subunit stoichiometry of the GroEL–Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.
Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular‐specific proteomic approach to identify rare intrinsic membrane proteins
Authors: Seigneurin‐Berny Daphné , Rolland Norbert , Garin Jérome , Joyard Jacques 
Published: March 2003

SummaryIdentification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane α‐helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS‐PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.
Protein dynamics, activity and cellular localization of soybean lipoxygenases indicate distinct functional roles for individual isoforms
Authors: Fischer Andreas M. , Dubbs Wesley E. , Baker Rachel A. , Fuller Mark A. , Stephenson Lowry C. , Grimes Howard D. 
Published: March 2003

SummaryVegetative lipoxygenases (VLXs) in soybean are hypothesized to function in nitrogen storage and partitioning. Isoform‐specific antibodies for four of the five known VLX isoenzymes were used to investigate the influence of source‐sink status on protein levels, as well as to analyze the tissue and subcellular localization of the different isoforms. VLXD responded most strongly to sink limitation, although the levels of VLXA, B and C increased as well. After sink limitation, VLXD and the vegetative storage protein, VSPα, accumulated in the vacuoles of bundle sheath and paraveinal mesophyll cells, while VLXA, B and C localized to the cytosol of these cells. All five known VLX isoenzymes were active with both linoleic and linolenic acid substrates after expression in Escherichia coli. The strong upregulation of VLXD levels after sink limitation as well as the localization of this isoform to the vacuoles of paraveinal mesophyll and bundle sheath cells (where VSPs are found) strongly suggest that VLXD should be considered as a major storage protein in soybean leaves. Furthermore, since VLXA, B and C also accumulate in sink‐limited soybean leaves, are located in the cytosol of paraveinal mesophyll cells and are active at pH values typically found in this compartment, their activities may well contribute to lipid metabolism in this tissue. This multi‐gene family is thus ideally poised to play a pivotal role in the balance of N deposition relative to lipid‐based storage, defense or signaling, by modulating contribu‐ tions to these processes in the transient storage cells of the paraveinal mesophyll.
HOS5–a negative regulator of osmotic stress‐induced gene expression in Arabidopsis thaliana
Authors: Xiong Liming , Ishitani Manabu , Lee Hojoung , Zhu Jian‐Kang 
Published: March 2003

SummaryOsmotic stress activates the expression of many plant genes through ABA‐dependent as well as ABA‐independent signaling pathways. We report here the characterization of a novel mutant of Arabidopsis thaliana, hos5‐1, which exhibits increased expression of the osmotic stress responsive RD29A gene. The expression of several other stress genes are also enhanced by the hos5‐1 mutation. The enhanced expression is specific to ABA and osmotic stress because low temperature regulation of these genes is not altered in the mutant. Genetic analysis indicated that hos5‐1 is a recessive mutation in a single nuclear gene on chromosome III. Double mutant analysis of hos5‐1 and the ABA‐deficient aba1‐1 as well as the ABA‐insensitive abi1‐1 mutant indicated that the osmotic stress hypersensitivity of hos5‐1 is not affected by ABA deficiency or insensitivity. Furthermore, combined treatments of hos5‐1 with ABA and osmotic stress had an additive effect on RD29A‐LUC expression. These results suggest that the osmotic stress hypersensitivity in hos5‐1 may be ABA‐independent. The germination of hos5‐1 seeds was more resistant to ABA. However, the hos5‐1 mutation did not influence stomatal control and only slightly affected the regulation of growth and proline accumulation by ABA. The hos5‐1 mutation reveals a negative regulator of osmotic stress‐responsive gene expression shared by ABA‐dependent and ABA‐independent osmotic stress signaling pathways.
Single pollen typing combined with laser‐mediated manipulation
Authors: Matsunaga Sachihiro , Schütze Karin , Donnison Iain S. , Grant Sarah R. , Kuroiwa Tsuneyoshi , Kawano Shigeyuki 
Published: March 2003

SummaryWe combined single pollen typing with laser‐mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV‐laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non‐contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer‐extension‐preamplification PCR (I‐PEP PCR). Nested PCR with sequence tagged site (STS)‐specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome‐linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.
Plant viral suppressors of post‐transcriptional silencing do not suppress transcriptional silencing
Authors: Marathe Rajendra , Smith Trenton H. , Anandalakshmi Radhamani , Bowman Lewis H. , Fagard Mathilde , Mourrain Philippe , Vaucheret Hervé , Vance Vicki B. 
Published: March 2003

SummaryHomology‐dependent gene silencing is a regulatory mechanism that limits RNA accumulation from affected loci either by suppression of transcription (transcriptional gene silencing, TGS) or by activation of a sequence‐specific RNA degradation process (post‐transcriptional gene silencing, PTGS). The P1/HC‐Pro sequence of plant potyviruses and the 2b gene of the cucumber mosaic virus have been shown to interfere with PTGS. The ability of these viral suppressors of PTGS to interfere with TGS was tested using the 271 locus which imposes TGS on transgenes under 35S or 19S promoters and PTGS on the endogenous nitrite reductase gene (Nii). Both P1/HC‐Pro and 2b reversed PTGS of Nii genes in 271‐containing tobacco plants, but failed to reverse TGS of 35S‐GUS transgenes in the same plant. P1/HC‐Pro expression from a transgene also failed to suppress either the initiation or maintenance of TGS imposed by the NOSpro‐silencing locus, H2. These results indicate that PTGS and TGS operate through unlinked pathways or that P1/HC‐Pro and 2b interfere at step(s) in PTGS that are downstream of any common components in the two pathways. The data suggest a simple assay to identify post‐transcriptionally silenced transgenic lines with the potential to be stably converted to high expressing lines.
A high throughput system for transposon tagging and promoter trapping in tomato
Authors: Meissner Rafael , Chague Veronique , Zhu Qianho , Emmanuel Eyal , Elkind Yonatan , Levy Avraham A. 
Published: March 2003

SummaryWe describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro‐Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds‐tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using β‐glucuronidase (GUS). We show that luciferase can be used as a non‐invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2–3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high‐throughput functional analysis of genes and promoter isolation in tomato.