bluuebunny/crossref_metadata_2025 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

Tomato (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

Maternally 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

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

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

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

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

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

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

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

Identification 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

Vegetative 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

Osmotic 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

We 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

Homology‐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

We 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.
Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis
Authors: Kawaoka Akiyoshi , Kaothien Pulla , Yoshida Kazuya , Endo Saori , Yamada Keiko , Ebinuma Hiroyasu
Published: March 2003

The AC‐rich motif, Pal‐box, is an important cis‐acting element for gene expression involved in phenylpropanoid biosynthesis. A cDNA clone (Ntlim1) encoding a Pal‐box binding protein was isolated by Southwestern screening. The deduced amino acid sequence is highly similar to the members of the LIM protein family that contain a zinc finger motif. Moreover, Ntlim1 had a specific DNA binding ability and transiently activated the transcription of a β‐glucuronidase reporter gene driven by the Pal‐box sequence in tobacco protoplasts. The transgenic tobacco plants with antisense Ntlim1 showed low levels of transcripts from some key phenylpropanoid pathway genes such as phenylalanine ammonia‐lyase, hydroxycinnamate CoA ligase and cinnamyl alcohol dehydrogenase. Furthermore, a 27% reduction of lignin content was observed in the transgenic tobacco with antisense Ntlim1.
Salicylic acid is not required for Cf‐2‐ and Cf‐9‐dependent resistance of tomato to Cladosporium fulvum
Authors: Brading Penny A. , Hammond‐Kosack Kim E. , Parr Adrian , Jones Jonathan D. G.
Published: March 2003

Tomato leaves or cotyledons expressing the Cf‐2 or Cf‐9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene‐dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild‐type Cf0. Neither free nor conjugated salicylic acid accumulated in IF‐infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf‐9‐dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf‐2‐like response. SA also potentiated Cf‐9‐mediated necrosis in IF‐infiltrated wild‐type leaves. In contrast, the Cf‐2‐dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild‐type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF‐induced accumulation of three SA‐inducible defence‐related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm‐22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV‐inoculated N gene‐containing NahG tobacco plants.
Molecular characterization of a novel glucose‐6‐phosphate dehydrogenase from potato (Solanum tuberosum L.)
Authors: Wendt Urte Kristina , Wenderoth Irina , Tegeler Achim , Von Schaewen Antje
Published: March 2003

We describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox‐regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C‐terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein‐mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid‐localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.
Transgenic potato plants reveal the indispensable role of cystathionine β‐lyase in plant growth and development
Authors: Maimann Stefanie , Wagner Cornelia , Kreft Oliver , Zeh Michaela , Willmitzer Lothar , Höfgen Rainer , Hesse Holger
Published: March 2003

Methionine (Met) is an essential amino acid that is often unavailable at sufficient dietary levels. In order to better understand Met pathway regulation, a cDNA encoding cystathionine β‐lyase (CbL; EC 4.4.1.8) has been cloned from Solanum tuberosum. An antisense construct of this gene was used to generate transgenic potato plants with reduced CbL levels. Transgenic plants exhibiting leaf CbL activity levels of up to 50% below wild‐type levels were obtained. Metabolite analysis revealed a reduction in Met levels in these CbL antisense plants, as well as remarkable increases in the pathway intermediates cystathionine, homoserine and cysteine. Unexpectedly, an increase in homocysteine was also observed. Levels of aspartate amino acid pathway intermediates (including aspartate, lysine and threonine) remained essentially unaffected. Neither transcript levels nor protein products of other pathway‐relevant genes were altered significantly in these plants. CbL antisense plants exhibited an altered phenotype characterized by a bushy growth habit, small light‐green leaves and small tubers. This phenotype could be alleviated upon Met supplementation, suggesting that low Met levels, rather than pathway intermediate accumulation, is responsible for the phenotypic effects of CbL transgene expression. These data unequivocally demonstrate the central role of CbL in Met biosynthesis, and, subsequently, in plant growth and development.
Maize ROP7 GTPase contains a unique, CaaX box‐independent plasma membrane targeting signal
Authors: Ivanchenko Maria , Vejlupkova Zuzana , Quatrano Ralph S. , Fowler John E.
Published: March 2003

Signals in the carboxy‐terminal hypervariable region (HVR) of Rho and Ras GTPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Rops (a plant‐specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C‐terminal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potential sites for post‐translational modification that leads to membrane association; one is in an internal CaaX motif, which would be at the C‐terminus if the final intron in both genes were not removed. Transient expression of a GFP–ROP7 fusion revealed its near‐total association with the plasma membrane (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Surprisingly, the cysteine in the terminal CAA is not required for PM targeting of GFP–ROP7. In contrast, an internal HVR cysteine is essential for proper targeting of the fusion, and the cysteine in the internal CaaX is required for complete membrane association. Interestingly, this CaaX motif can also direct PM association when placed at the fusion C‐terminus by addition of an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that the ROP7 HVR directs PM localization by a mechanism independent of a C‐terminal CaaX motif; this mechanism may have evolved through addition of 3′ intron/exon sequences to aropprogenitor.
Cytokinin and gibberellin activate SaMADS A, a gene apparently involved in regulation of the floral transition in Sinapis alba
Authors: Bonhomme Françoise , Kurz Birgit , Melzer Siegbert , Bernier Georges , Jacqmard Annie
Published: March 2003

In plants of Sinapis alba induced to flower by one long day, the MADS box gene, SaMADS A, is expressed initially in the central corpus (L3 cells) of the shoot apical meristem (SAM), about 1.5–2 days before initiation of the first floral meristem. We have combined a physiological approach by testing the effects of three putative floral signals on SaMADS A expression in the SAM of S. alba plants with a transgenic approach using Arabidopsis thaliana plants. A single application of a low dose of a cytokinin or a gibberellin to the apex of vegetative S. alba plants is capable of mimicking perfectly the initial effect of the long day on SaMADS A transcription. A treatment combining the two hormones causes the same activation but seems to enhance the level of SaMADS A expression. A sucrose application to the apex of vegetative plants is, on the contrary, unable to activate SaMADS A expression. None of these chemicals, alone or combined, is capable of causing the floral shift at the SAM. Since the constitutive expression of SaMADS A leads to precocious flowering in A. thaliana and antisense expression of a fragment of the A. thaliana homologue AGL20 leads to a delay in flowering time, these results are consistent with SaMADS A activation being an intermediate event in a cytokinin‐ and/or gibberellin‐triggered signal transduction pathway that is involved in the regulation of floral transition in S. alba.
Construction of male and female PAC genomic libraries suitable for identification of Y‐chromosome‐specific clones from the liverwort, Marchantia polymorpha
Authors: Okada Sachiko , Fujisawa Masaki , Sone Takefumi , Nakayama Shigeki , Nishiyama Rie , Takenaka Mizuki , Yamaoka Shohei , Sakaida Megumi , Kono Kaoru , Takahama Masayoshi , Yamato Katsuyuki T. , Fukuzawa Hideya , Brennicke Axel , Ohyama Kanji
Published: March 2003

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1‐derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male‐specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male‐specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.
Tonoplast subcellular localization of maize cytochrome b5 reductases
Authors: Bagnaresi Paolo , Mazars‐Marty Daniele , Pupillo Paolo , Marty Francis , Briat Jean‐François
Published: March 2003

Plant cytochrome b5 reductases (b5R) are assumed to be part of an ER‐associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b5) to ER‐associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem. J. 338, 499–505). Maize b5R isoforms are produced by a small multi‐gene family. The NFR cDNAs were shown to encode active b5Rs by heterologous expression in yeast. Both reductases, in addition to Fe3+‐chelates, efficiently reduced Cu2+‐chelates. Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots. Unexpectedly, maize b5Rs were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast‐enriched maize root fractions. Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b5R. This indicates that alternative ER‐associated electron donors for desaturases need to be sought, and that plant b5Rs may have previously unexpected functions.
The MLA6 coiled‐coil, NBS‐LRR protein confers AvrMla6‐dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat
Authors: Halterman Dennis , Zhou Fasong , Wei Fusheng , Wise Roger P. , Schulze‐Lefert Paul
Published: March 2003

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla‐RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full‐length CC‐NBS‐LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full‐length candidate cDNA, two single‐cell expression assays were used to demonstrate complementation of AvrMla6‐dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1‐independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC‐NBS‐LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.
Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate
Authors: Richardson Alan E. , Hadobas Paul A. , Hayes Julie E.
Published: March 2003

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild‐type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20‐fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.
The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily
Authors: Swiderski Michal R. , Innes Roger W.
Published: March 2003

Specific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1. Previous work has shown that RPS5 encodes a member of the nucleotide binding site‐leucine rich repeat class of plant disease resistance genes. Here we report that PBS1 encodes a putative serine‐threonine kinase. Southern blot analysis revealed that the pbs1‐1 allele contained a deletion of the 3′ end of the PBS1 open reading frame. DNA sequence analysis of the pbs1‐2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases. The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants. Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function. The Pto kinase of tomato, which is required for specific resistance to P. syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain. These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins. We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition.
Phosphatidic acid activates a wound‐activated MAPK in Glycine max
Authors: Lee Sumin , Hirt Heribert , Lee Youngsook
Published: March 2003

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound‐activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n‐butanol, an inhibitor of phospholipase D, the wound‐induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension‐cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA‐induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound‐induced MAPK signaling in plants.
Amino acid permeases in developing seeds ofVicia fabaL.: expression precedes storage protein synthesis and is regulated by amino acid supply
Authors: Miranda Manoela , Borisjuk Ljudmilla , Tewes Annegret , Heim Ute , Sauer Norbert , Wobus Ulrich , Weber Hans
Published: March 2003

Full length cDNAs encoding three amino acid permeases were isolated from seed‐specific libraries ofVicia faba. The predicted proteins VfAAP1, VfAAP3 and VfAAP4 share up to 66% identity among themselves. Functional characterization of VfAAP1 and VfAAP3 in a yeast mutant showed that these permeases transport a broad range of amino acids. However, VfAAP1 had a preference for cysteine and VfAAP3 for lysine and arginine.VfAAP1was highly expressed in cotyledons at early developmental stages and moderately in other sink tissues. Its peak of expression in cotyledons corresponded to the appearance of storage protein transcripts, suggesting that this transporter fulfills an important role in providing amino acids for storage protein biosynthesis.VfAAP3was expressed most abundantly in maternal tissues, that is in roots, stems, gynoecia, pods and seed coats at different developmental stages.VfAAP4transcripts could not be detected by northern hybridization.In situhybridization showed thatVfAAP1mRNA is distributed throughout cotyledon storage parenchyma cells, but could not be detected in the abaxial epidermal cell layer. It also accumulate in the chlorenchyma and thin‐walled parenchyma cells of seed coats.VfAAP1mRNA levels were lower in cotyledons cultured in the presence of glutamine, whereas expression of a vicilin storage protein gene was up‐regulated under similar conditions. Cysteine repressed the expression of theGUSreporter gene under control of theVfAAP1promoter, suggesting that this transporter is modulated at the transcriptional level. Regulation of amino acid transport in relation to storage protein accumulation is discussed.
A plastid envelope location of Arabidopsis ent‐kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway
Authors: Helliwell Chris A. , Sullivan James A. , Mould Ruth M. , Gray John C. , Peacock W. James , Dennis Elizabeth S.
Published: March 2003

We have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono‐oxygenase and 2‐oxoglutarate‐dependent dioxygenase classes. We show that the N‐terminal pre‐sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent‐kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1–GFP fusion is also imported by isolated pea chloroplasts. The N‐terminal portion of the cytochrome P450 protein ent‐kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S‐labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent‐kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid‐ and endoplasmic reticulum‐located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.
Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions
Authors: Hilpert Bernadette , Bohlmann Holger , Den Camp Roel op , Przybyla Dominika , Miersch Otto , Buchala Anthony , Apel Klaus
Published: March 2003

Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin‐like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA‐dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet‐mutants have been isolated which showed a constitutive expression of the thionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1‐GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12‐oxophytodienoic acid (OPDA) up to 40‐fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)‐ and the JA‐dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet‐mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.