bluuebunny/crossref_metadata_2025 huggingface dataset:
Translational control in plant stress: the formation of messenger ribonucleoprotein particles (mRNPs) in response to desiccation of Tortula ruralis gametophytes
Authors: Wood Andrew J. , Oliver Melvin J.
Published: March 2003

Changes in gene expression observed in vivo in response to desiccation and rehydration of the desiccation‐tolerant bryophyte Tortula ruralis are regulated by alterations in the pattern of selection of messenger RNAs, from a qualitatively constant mRNA pool, by the translational machinery. When drying rates are slow, messenger ribonucleoprotein particles (mRNPs) are formed in the drying gametophytes. A representative rehydrin mRNA, Tr288, was sequestered into these particles which were analysed using sucrose and CsCl gradients. Quantitative RT–PCR analysis of the fractions from a low salt extraction demonstrated that Tr288 mRNA migrated farther in the sucrose gradient, relative to those extractable in high salt, indicating that the transcript is associated with particles that are of higher density. RT–PCR analysis also demonstrated that the majority of Tr288 mRNA, from slowly desiccated gametophytes, is associated with particles that have buoyant densities between 1.44 and 1.64 g cm– 3 which correspond to the buoyant density range reported for mRNP particles. mRNPs that are unique to drying T. ruralis gametophytes form at least four size classes after in vivo UV cross‐linking based upon FPLC analysis. This is the first report of mRNP formation in response to a vegetative water deficit in plants.
Temperature sensing by plants: the primary characteristics of signal perception and calcium response
Authors: Plieth Christoph , Hansen Ulf‐Peter , Knight Heather , Knight Marc R.
Published: March 2003

Cold elicits an immediate rise in the cytosolic free calcium concentration ([Ca

2+

]

c

) of plant cells. We have studied the concerted action of the three underlying mechanisms, namely sensing, sensitisation and desensitisation, which become important when plants in the field are subjected to changes in temperature. We applied different regimes of temperature changes with well‐defined cooling rates to intact roots of Arabidopsis thaliana expressing the calcium‐indicator, aequorin. Our results indicate that temperature sensing is mainly dependent on the cooling rate, dT/dt, whereas the absolute temperature T is of less importance. Arabidopsis roots were found to be sensitive to cooling rates of less than dT/dt = 0.01°C/s. However, at cooling rates below 0.003°C/s (i.e. cooling 10°C in 1 h) there is no detectable [Ca

2+

]

c

response at all. At low temperature, the sensitivity of the plant cold‐detection system is increased. This in turn produces greater cooling‐induced [Ca

2+

]

c

elevations. Prolonged or repeated cold treatment attenuates the [Ca

2+

]

c

responses to subsequent episodes of cooling.
Leaf C40.4: a carotenoid‐associated protein involved in the modulation of photosynthetic efficiency?
Authors: Monte Elena , Ludevid Dolors , Prat Salomé
Published: March 2003

Comparative analysis by differential RNA display (DDRT–PCR) of the expression patterns of potato plants induced and non‐induced to tuberize, led to the isolation of a cDNA clone, C40.4, that is strongly upregulated in the leaves of tuberizing plants. Leaf expression of this transcript was shown to be light‐dependent, with increased levels of mRNA and protein being detected during the light hours. Sequence analysis revealed near complete identity to potato CDSP34, a thylakoid protein induced by drought stress, and strong homology to the carotenoid‐associated proteins fibrillin, ChrB and PAP from pepper fruit, and CHRC from cucumber flowers. By using an antibody against pepper fibrillin, we were able to demonstrate a chloroplastic location of the C40.4 protein in association with the thylakoid membranes. Two‐dimensional analysis of thylakoid pigment–protein complexes showed a specific association of the protein with the photosystem II (PSII) multi‐subunit complex. Antisense plants with reduced levels of accumulation of C40.4 showed a stunted growth and decreased tuber yield and exhibited reduced values of non‐photochemical quenching of chlorophyll a fluorescence. Altogether, these results indicate a preferential association of the C40.4 protein with the light harvesting complex of photosystem II (LHCII) antenna pigment complexes, and suggest a functional role of this protein in photosynthesis, by modulating photosynthetic efficiency and dissipation of excess absorbed light energy within the antenna complex.
In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency
Authors: Eibl Christian , Zou Zhurong , Beck Andreas , Kim Minkyun , Mullet John , Koop Hans‐Ulrich
Published: March 2003

5′ and 3′ untranslated regions (UTRs) of plastid RNAs act as regulatory elements for post‐transcriptional control of gene expression. Polyethylene glycol‐mediated plastid transformation with UTR–GUS reporter gene fusions was used to study the function of the psbA, rbcL and rpl32 UTRs in vivo. All gene fusions were expressed from the same promoter, i.e. the promoter of the 16S‐rRNA gene, such that variations in RNA and protein levels would be due to the involved UTR elements alone. Transgenic tobacco lines containing different combinations of UTRs showed fivefold variation in the uidA–mRNA level (RNA stability) and approximately 100‐fold differences in GUS activity, a measure of translation activity. The rbcL 5′‐UTR conferred greater mRNA stability than the psbA 5′‐UTR on uidA transcripts. In contrast, the psbA 5′‐UTR enhanced translation of GUS to a much greater extent compared to the rbcL 5′‐UTR. The psbA 5′‐UTR also mediated light‐induced activation of translation which was not observed with other constructs. Deletion mutagenesis of an unanalysed terminal sequence element of the psbA 5′‐UTR resulted in a twofold drop in uidA‐mRNA level and a fourfold decrease in translation efficiency. Exchange of 3′‐UTRs results in up to fivefold changes of mRNA levels and does not significantly influence translation efficiency. The mechanical impacts of these results on plastid translation regulation are discussed.
A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions
Authors: Del Pozo Juan Carlos , Allona Isabel , Rubio Vicente , Leyva Antonio , De La Peña Alicia , Aragoncillo Cipriano , Paz‐Ares Javier
Published: March 2003

Low phosphorous availability, a common condition of many soils, is known to stimulate phosphatase activity in plants; however, the molecular details of this response remain mostly unknown. We purified and sequenced the N‐terminal region of a phosphate starvation induced acid phosphatase (AtACP5) from Arabidopsis thaliana, and cloned its cDNA and the corresponding genomic DNA. The nucleotide sequence of the cDNA predicted that AtACP5 is synthesised as a 338 amino acid‐long precursor with a signal peptide. AtACP5 was found to be related to known purple acid phosphatases, especially to mammal type 5 acid phosphatases. Other similarities with purple acid phosphatases, which contain a dinuclear metal centre, include the conservation of all residues involved in metal ligand binding and resistance to tartrate inhibition. In addition, AtACP5, like other type 5 acid phosphatases, displayed peroxidation activity. Northern hybridisation experiments, as well as in situ glucuronidase (GUS) activity assays on transgenic plants harbouring AtACP5:GUS translational fusions, showed that AtACP5 is not only responsive to phosphate starvation but also to ABA and salt stress. It is also expressed in senescent leaves and during oxidative stress induced by H2O2, but not by paraquat or salicylic acid. Given its bifunctionality, as it displays both phosphatase and peroxidation activity, we propose that AtACP5 could be involved in phosphate mobilisation and in the metabolism of reactive oxygen species in stressed or senescent parts of the plant.
Hairpin elements, the first family of foldback transposons (FTs) in Arabidopsis thaliana
Authors: Adé Jules , Belzile François J.
Published: March 2003

We report here on the identification in Arabidopsis thaliana of a new family of transposable elements named Hairpin. These elements are related to foldback transposons (FTs), a large and heterogeneous group of transposable elements first described in Drosophila and recently in Solanaceae. Hairpin elements are the first family of FTs reported in Arabidopsis thaliana and the first family of FTs of type 3 to be described in the plant kingdom. In contrast to previous FTs described, Hairpin appears to be a homogeneous family in size (238 ± 7 bp) as well as in structure. Hairpin elements are dispersed in the Arabidopsis genome and Southern hybridization revealed that they are present in relatively low copy numbers. Finally, we discuss the potential usefulness of these elements in studying the phylogenetic relationship between Arabidopsis ecotypes.
A family of antimicrobial peptides is produced by processing of a 7S globulin protein in Macadamia integrifolia kernels
Authors: Marcus John P. , Green Jodie L. , Goulter Ken C. , Manners John M.
Published: March 2003

A new family of antimicrobial peptides has been discovered in Macadamia integrifolia. The first member of this new family to be purified from nut kernels was a peptide of 45 aa residues, termed MiAMP2c. This peptide inhibited various plant pathogenic fungi in vitro. cDNA clones corresponding to MiAMP2c encoded a 666 aa precursor protein homologous to vicilin 7S globulin proteins. The deduced precursor protein sequence contained a putative hydrophobic N‐terminal signal sequence (28 aa), an extremely hydrophilic N‐proximal region (212 aa), and a C‐terminal region of 426 aa which is represented in all vicilins. The hydrophilic portion of the deduced protein contained the sequence for MiAMP2c as well as three additional segments having the same cysteine spacing pattern as MiAMP2c. Each member of the MiAMP2 family (i.e. MiAMP2a, b, c and d) consisted of approximately 50 amino acids and contained a C‐X‐X‐X‐C‐(10–12)X‐C‐X‐X‐X‐C motif. Sub‐ sequent isolations from seed exudates led to the purification of the predicted family members MiAMP2b and 2d, both of which also exhibited antimicrobial activity in vitro. These results suggest that some vicilins play a role in defence during seed germination.
A novel myb oncogene homologue in Arabidopsis thaliana related to hypersensitive cell death
Authors: Daniel Xavier , Lacomme Christophe , Morel Jean‐Benoît , Roby Dominique
Published: March 2003

A novel myb oncogene homologue (AtMYB30) has been isolated by differential screening of a cDNA library prepared from Xanthomonas campestris pv. campestris (X. campestris)‐inoculated Arabidopsis thaliana cells cultured in the presence of cycloheximide. AtMYB30 is a single‐copy gene, and the encoded protein contains a MYB domain highly homologous to other plant and animal MYB proteins. Analyses of transcript levels in A. thaliana plants, or in cultured A. thaliana cells infected with either virulent or avirulent strains of the pathogens X. campestris and Pseudomonas syringae pv. tomato, showed that maximal levels of transcription of this gene occurred during the hypersensitive response. Furthermore, in A. thaliana mutants affected in the control of cell death initiation (lsd3, lsd4 and lsd5), constitutive expression or expression in lesion‐positive plants was observed, while in suppressors of the mutations lsd5 and lsd4, AtMYB30 transcripts did not accumulate. However, AtMYB30 expression could not be detected in the lsd1 mutant, which was hyper‐responsive to cell death initiators and unable to limit the extent of cell death, whatever the environmental conditions. The results presented here suggest a strong correlation between AtMYB30 and genetically controlled cell death, with a role in the initiation of cell death rather than in the limitation of its extent. Our results further indicate that the lsd mutants constitute an appropriate genetic model for studying the role of this gene in hypersensitive cell death, and their relation to different steps of the pathway(s) leading to cell death.
A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis
Authors: Martínez‐García Jaime F. , Monte Elena , Quail Peter H.
Published: March 2003

Although Arabidopsis has numerous well documented advantages for genetic and molecular analyses, its small size can be a limitation for biochemical and immunochemical assays requiring protein extraction. We have developed a rapid method to extract total protein from small amounts of Arabidopsis tissue that can be used for quantitative immunoblot analysis. The procedure involves direct extraction of tissue into SDS‐containing buffer under conditions permitting immediate protein quantification in the extract, using commercially available kits without prior fractionation. This approach provides maximal extraction and quantitative recovery of total cellular protein, together with accurate evaluation of target protein levels as a proportion of the total. We have examined the utility and sensitivity of the procedure using monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high‐ and low‐abundance members, respectively, of the phytochrome family in Arabidopsis. Both phytochromes could be rapidly and readily quantified in the tissues examined, with phyC being detectable in extracts representing as few as five dark‐grown seedlings, two light‐grown seedlings, or half a single leaf from 3‐week‐old adult plants. The data indicate that the procedure may have broad utility for the detection and quantitative analysis of many proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis phyC‐overexpressor seedlings to demonstrate that the procedure can be used to detect transgene‐encoded protein early at the segregating T2 generation, thereby offering the capacity for accelerated screening and selection of lines engineered to overexpress target proteins.
A tomato homeobox gene (HD‐Zip) is involved in limiting the spread of programmed cell death
Authors: Mayda Esther , Tornero Pablo , Conejero Vicente , Vera Pablo
Published: March 2003

Antisense suppression in transgenic tomato plants ofH52, a gene encoding a new homeodomain protein of the HD‐Zip class, produces a conditional lethal phenotype. The transgenic lines that survive exhibit spontaneous misregulation of cell death control in leaves, which, once initiated, propagates and engulfs the entire leaf. Activation of defence genes, over‐accumulation of ethylene and conjugated salicylic acid, and growth reduction of virulent pathogens also occurs in these plants. In wild‐type plants,H52is up‐regulated upon infection, mirroring the generation of the oxidative burst which normally precedes the hypersensitive response (HR). Thus, H52 appears to be a transcription factor involved in cellular protection by limiting spread of programmed cell death in plants.
Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine‐rich nuclear export signals in Arabidopsis thaliana
Authors: Haasen Dorothea , Köhler Claudia , Neuhaus Gunther , Merkle Thomas
Published: March 2003

Transport across the nuclear envelope is mediated by transport receptors from the Importin β family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine‐rich nuclear export signals (NESs). AtXPO1 shares 42–50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two‐hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine‐rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY‐2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.
Immunological analysis of the phosphorylation state of maize C4‐form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide
Authors: Ueno Yoshihisa , Imanari Eiji , Emura Junji , Yoshizawa‐Kumagaye Kumiko , Nakajima Kiichiro , Inami Kaoru , Shiba Tetsuo , Sakakibara Hitoshi , Sugiyama Tatsuo , Izui Katsura
Published: March 2003

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme’s sensitivity to its feedback inhibitor, L‐malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state‐specific antibodies to the C4‐form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15‐mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity‐chromatography. Specificity of the resulting antibodies to the C4‐form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root‐form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi‐quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13‐week‐old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid‐morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4‐form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen‐limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.
Post‐transcriptional defects in tobacco chloroplast mutants lacking the cytochrome b6/f complex
Authors: Monde Rita‐Ann , Zito Francesca , Olive Jacqueline , Wollman Francis‐André , Stern David B.
Published: March 2003

A variety of post‐transcriptional mechanisms govern the synthesis and assembly of photosynthetic protein complexes in chloroplasts. To test whether such mechanisms are conserved between photosynthetic algae and vascular plants, we have interrupted the chloroplast petA, petB and petD genes of tobacco, which encode three subunits of the cytochrome b6/f complex, and compared our results to those previously obtained with Chlamydomonas reinhardtii. As expected, the mutants exhibited high chlorophyll fluorescence, consistent with the loss of a functional cytochrome b6/f complex. Unlike the corresponding mutants of Chlamydomonas, however, cytochrome f was barely detectable in the ΔpetB or ΔpetD mutants. The amounts of petB‐ and petD‐containing mRNAs were reduced in the mutants compared to wild‐type plants, but the remaining mRNA was normally associated with polysomes. In contrast, there was a decrease in polysome association of the polycistronic petA mRNA in the ΔpetB and ΔpetD mutants, suggesting that the synthesis of cytochrome f may be decreased in the absence of cytochrome b6 or SUIV. These results are discussed in light of the translational autoregulation model that has been proposed for cytochrome b6/f complex assembly in Chlamydomonas.
Lesions in the sterol Δ7 reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis
Authors: Choe Sunghwa , Tanaka Atsushi , Noguchi Takahiro , Fujioka Shozo , Takatsuto Suguru , Ross Amanda S. , Tax Frans E. , Yoshida Shigeo , Feldmann Kenneth A.
Published: March 2003

The brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Δ7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark‐green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C‐labeled precursors in dwf5 verified a deficiency in a sterol Δ7 reductase activity. All six independent alleles contain loss‐of‐function mutations in the sterol Δ7 reductase gene. These include a putative mRNA instability mutation in dwf5‐1, 3′ and 5′ splice‐site mutations in dwf5‐2 and dwf5‐6, respectively, premature stop codons in dwf5‐3 (R400Z) and dwf5‐5 (R409Z), and a mis‐sense mutation in dwf5‐4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild‐type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith–Lemli–Opitz syndrome are caused by loss‐of‐function mutations in a sterol Δ7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.
Heat‐shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T‐DNA insertions
Authors: Matsuhara Shio , Jingu Fumito , Takahashi Taku , Komeda Yoshibumi
Published: March 2003

This paper describes expression profiles of the Arabidopsis HSP18.2 heat‐shock gene promoter by using three different reporter genes, and the application of this promoter to a method we have developed to drive heat‐shock‐dependent transcription of plant genome DNA flanked by T‐DNA insertions. We show that, irrespective of the location of the T‐DNA insertion, an HSP18.2 promoter towards the left border of the T‐DNA effectively induces transcription of flanking genome sequences in response to heat shock. If polyadenylated, tagged transcripts can be easily identified by RT–PCR.
Heterotrimeric G‐proteins of a filamentous fungus regulate cell wall composition and susceptibility to a plant PR‐5 protein
Authors: Coca María A. , Damsz Barbara , Yun Dae‐Jin , Hasegawa Paul M. , Bressan Ray A. , Narasimhan Meena L.
Published: March 2003

Membrane permeabilizing plant defensive proteins first encounter the fungal cell wall that can harbor specific components that facilitate or prevent access to the plasma membrane. However, signal transduction pathways controlling cell wall composition in filamentous fungi are largely unknown. We report here that the deposition of cell wall constituents that block the action of osmotin (PR‐5), an antifungal plant defense protein, against Aspergillus nidulans requires the activity of a heterotrimeric G‐protein mediated signaling pathway. The guanidine nucleotide GDPβS, that locks G‐proteins in a GDP‐bound inactive form, inhibits osmotin‐induced conidial lysis. A dominant interfering mutation in FadA, the α‐subunit of a heterotrimeric G‐protein, confers resistance to osmotin. A deletion mutation in SfaD, the β‐subunit of a heterotrimeric G‐protein also increases osmotin resistance. Aspergillus nidulans strains bearing these mutations also have increased tolerance to SDS, reduced cell wall porosity and increased chitin content in the cell wall.
Isolation and characterization of HsfA3, a new heat stress transcription factor of Lycopersicon peruvianum
Authors: Bharti Kapil , Schmidt Enrico , Lyck Ruth , Heerklotz Dirk , Bublak Daniela , Scharf Klaus‐Dieter
Published: March 2003

Stress‐induced transcription of heat shock proteins (Hsps) in eukaryotes is mediated by a conserved class of transcription factors called heat stress transcription factors (Hsfs). Here we report the isolation and functional characterization of HsfA3, a new member of the Hsf family. HsfA3 was cloned from a tomato heat stress cDNA library by yeast two‐hybrid screening, using HsfA1 as a bait. HsfA3 is a single‐copy gene with all the conserved sequence elements characteristic of a heat stress transcription factor. The constitutively expressed HsfA3 is mainly found in the cytoplasm under control conditions and in the nucleus under heat stress conditions. Functionally, HsfA3 behaves similarly to the already known members of tomato Hsf family. It is able to substitute yeast Hsf for viability functions and is a strong activator of Hsf‐dependent reporter constructs both in tobacco protoplasts and yeast. Finally, similar to the AHA motifs in HsfA1 and HsfA2, the activator function depends on four short peptide motifs with a central tryptophan residue found in the C‐terminal domain of HsfA3.
T‐DNA insertional mutagenesis for functional genomics in rice
Authors: Jeon Jong‐Seong , Lee Sichul , Jung Ki‐Hong , Jun Sung‐Hoon , Jeong Dong‐Hoon , Lee Jinwon , Kim Chanhong , Jang Seonghoe , Lee Shinyoung , Yang Kiyoung , Nam Jongmin , An Kyungsook , Han Min‐Jung , Sung Ryo‐Jin , Choi Hyun‐Sook , Yu Jung‐Hwa , Choi Jung‐Hwan , Cho Se‐Yu , Cha Sang‐Su , Kim Shi‐In , An Gynheung
Published: March 2003

We have produced 22 090 primary transgenic rice plants that carry a T‐DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel‐blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T‐DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T‐DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless β‐glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T‐DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6–2.1% of tested organs were GUS‐positive in the tested organs, and that their GUS expression patterns were organ‐ or tissue‐specific or ubiquitous in all parts of the plant. The large population of T‐DNA‐tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.
Simultaneous analysis of metabolites in potato tuber by gas chromatography–mass spectrometry
Authors: Roessner Ute , Wagner Cornelia , Kopka Joachim , Trethewey Richard N. , Willmitzer Lothar
Published: March 2003

A new method is presented in which gas chromatography coupled to mass spectrometry (GC–MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC–MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70–140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil‐ and in vitro‐grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.
Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana
Authors: Cheng Ning‐Hui , Su Chun‐Lin , Carter Shelly A. , Nelson Richard S.
Published: March 2003

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non‐vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non‐vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem‐associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem‐associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular‐associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.
Knockout of the regulatory site of 3‐ketoacyl‐ACP synthase III enhances short‐ and medium‐chain acyl‐ACP synthesis
Authors: Abbadi Amine , Brummel Monika , Spener Friedrich
Published: March 2003

3‐ketoacyl‐acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl‐acyl carrier protein (ACP) as substrates. Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium‐chain acyl‐ACP end products of the FAS reaction, i.e. inhibition by lauroyl‐ACP was uncompetitive towards acetyl CoA and non‐competitive with regard to malonyl‐ACP. This indicated a distinct attachment site for regulatory acyl‐ACPs. Based on alignment of primary structures of various KAS IIIs and 3‐ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl‐ACPs. Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity. Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl‐ACPs. To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium‐chain fatty acid‐producing Cuphea lanceolata seeds and long‐chain fatty acid‐producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant. High amounts of short‐chain acyl‐ACPs in the case of C. lanceolata, and of medium‐chain acyl‐ACPs in the case of rape seed preparations, were obtained. This approach targets regulation and offers new possibilities to derive transgenic or non‐transgenic plants for production of seed oils with new qualities.
Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose‐1,6‐bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana
Authors: Strand Åsa , Zrenner Rita , Trevanion Stephen , Stitt Mark , Gustafsson Petter , Gardeström Per
Published: March 2003

Photosynthetic carbon metabolism was investigated in antisense Arabidopsis lines with decreased expression of sucrose phosphate synthase (SPS) and cytosolic fructose‐1,6‐bisphosphatase (cFBPase). In the light, triose phosphates are exported from the chloroplast and converted to sucrose via cFBPase and SPS. At night, starch is degraded to glucose, exported and converted to sucrose via SPS. cFBPase therefore lies upstream and SPS downstream of the point at which the pathways for sucrose synthesis in the day and night converge. Decreased cFBPase expression led to inhibition of sucrose synthesis; accumulation of phosphorylated intermediates; Pi‐limitation of photosynthesis; and stimulation of starch synthesis. The starch was degraded to maintain higher levels of sugars and a higher rate of sucrose export during the night. This resembles the response in other species when expression of enzymes in the upper part of the sucrose biosynthesis pathway is reduced. Decreased expression of SPS inhibited sucrose synthesis, but phosphorylated intermediates did not accumulate and carbon partitioning was not redirected towards starch. Sugar levels and sucrose export was decreased during the night as well as during the day. Although ribulose‐1,5‐bisphosphate regeneration and photosynthesis were inhibited, the PGA/triose‐P ratio remained low and the ATP/ADP ratio high, showing that photosynthesis was not limited by the rate at which Pi was recycled during end‐product synthesis. Two novel responses counteracted the decrease in SPS expression and explain why phosphorylated intermediates did not accumulate, and why allocation was not altered in the antisense SPS lines. Firstly, a threefold decrease of PPi and a shift of the UDP‐glucose/hexose phosphate ratio favoured sucrose synthesis and prevented the accumulation of phosphorylated intermediates. Secondly, there was no increase of AGPase activity relative to cFBPase activity, which would prevent a shift in carbon allocation towards starch synthesis. These responses are presumably triggered when sucrose synthesis is decreased in the night, as well as by day.
Arabidopsis DNA ligase IV is induced by γ‐irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4
Authors: West Christopher E. , Waterworth Wanda M. , Jiang Qing , Bray Clifford M.
Published: March 2003

Rejoining of single‐ and double‐strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double‐strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by γ‐irradiation, represent one of the most serious threats to genomic integrity. Non‐homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP‐dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino‐acid sequence to the human DNA ligase IV. Yeast two‐hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV‐binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C‐terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by γ‐irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.
Tissue‐specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles
Authors: Hause Bettina , Stenzel Irene , Miersch Otto , Maucher Helmut , Kramell Robert , Ziegler Jöörg , Wasternack Claus
Published: March 2003

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor,cis(+)12‐oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue‐specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue‐specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin‐mediated wound response of tomato.
Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin‐binding isoform
Authors: Snedden Wayne A. , Blumwald Eduardo
Published: March 2003

Calmodulin is a regulatory protein activated during Ca2+ signalling. We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin‐binding diacylglycerol kinase) encoding a novel calmodulin‐binding protein with sequence similarity to diacylglycerol kinases from animals. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid. We delineated the calmodulin‐binding domain to approximately 25 residues near the C‐terminus of LeCBDGK. We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin‐binding domain. Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro. Anti‐DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions. The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin–agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin‐binding protein. In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions. Native LeCBDGK and calmodulin co‐immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo. The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin‐binding domain of LeCBDGK is encoded by a separate exon. Thus, alternative transcript splicing leads to calmodulin‐binding and non‐binding forms of diacylglycerol kinases in tomato. Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.
AtFim1 is an actin filament crosslinking protein from Arabidopsis thaliana
Authors: Kovar David R. , Staiger Christopher J. , Weaver Eric A. , McCurdy David W.
Published: March 2003

ATFIM1 is a widely expressed gene in Arabidopsis thaliana that encodes a putative actin filament‐crosslinking protein, AtFim1, belonging to the fimbrin/plastin class of actin‐binding proteins. In this report we have used bacterially expressed AtFim1 and actin isolated from Zea mays pollen to demonstrate that AtFim1 functions as an actin filament‐crosslinking protein. AtFim1 binds pollen actin filaments (F‐actin) in a calcium‐independent manner, with an average dissociation constant (Kd) of 0.55 ± 0.21 µM and with a stoichiometry at saturation of 1 : 4 (mol AtFim1 : mol actin monomer). AtFim1 also crosslinks pollen F‐actin by a calcium‐independent mechanism, in contrast to crosslinking of plant actin by human T‐plastin, a known calcium‐sensitive actin‐crosslinking protein. When micro‐injected at high concentration into living Tradescantia virginiana stamen hair cells, AtFim1 caused cessation of both cytoplasmic streaming and transvacuolar strand dynamics within 2–4 min. Using the ‘nuclear displacement assay’ as a measure of the integrity of the actin cytoskeleton in living stamen hair cells, we demonstrated that AtFim1 protects actin filaments in these cells from Z. mays profilin (ZmPRO5)‐induced depolymerization, in a dose‐dependent manner. The apparent ability of AtFim1 to protect actin filaments in vivo from profilin‐mediated depolymerization was confirmed by in vitro sedimentation assays. Our results indicate that AtFim1 is a calcium‐independent, actin filament‐crosslinking protein that interacts with the actin cytoskeleton in living plant cells.
Plastid‐expressed 5‐enolpyruvylshikimate‐3‐phosphate synthase genes provide high level glyphosate tolerance in tobacco
Authors: Ye Guang‐Ning , Hajdukiewicz Peter T.J. , Broyles Debra , Rodriguez Damian , Xu Charles W. , Nehra Narender , Staub Jeffrey M.
Published: March 2003

Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high‐level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid‐encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high‐level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over‐expression in plastids of tolerant forms of 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS). Immunological, enzymatic and whole‐plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10 000‐fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid‐expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid‐expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear‐encoded gene. Nevertheless, the demonstration of high‐level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.
Negative autoregulation of the Arabidopsis homeobox gene ATHB‐2
Authors: Ohgishi Maki , Oka Atsuhiro , Morelli Giorgio , Ruberti Ida , Aoyama Takashi
Published: March 2003

The Arabidopsis homeobox gene ATHB‐2 is tightly regulated by light signals, and is thought to direct morphological changes during shade avoidance responses. To understand how ATHB‐2 mediates light signals in plant morphogenesis, we investigated its transcriptional network. We constructed a gene encoding a chimeric transcription factor (HD‐Zip‐2‐V‐G) that is expected to activate target genes of ATHB‐2 in a glucocorticoid‐dependent manner. In transgenic Arabidopsis plants expressing HD‐Zip‐2‐V‐G, glucocorticoid treatment activates the ATHB‐2 gene itself, independent of de novo protein synthesis. An in vitro DNase I‐footprinting experiment showed that recombinant ATHB‐2 protein specifically bound to an ATHB‐2 promoter region. These complementary results indicate that ATHB‐2 recognizes its own promoter. Consistent with the fact that ATHB‐2 itself has been shown to act as a repressor, expression of the endogenous ATHB‐2 gene was repressed in transgenic plants overexpressing an ATHB‐2 transgene. Moreover, target‐gene analysis using the HD‐Zip‐2‐V‐G suggested that ATHB‐2 recognizes other HD‐Zip II subfamily genes. We conclude that ATHB‐2 has a negative autoregulatory loop and may be involved in a complicated transcriptional network involving paralogous genes, as is the case with animal homeobox genes.
Analysis of the function of two circadian‐regulated CONSTANS‐LIKE genes
Authors: Ledger Susan , Strayer Carl , Ashton Fern , Kay Steve A. , Putterill Jo
Published: March 2003

The Arabidopsis genes CONSTANS‐LIKE 1 (COL1) and CONSTANS‐LIKE 2 (COL2) are predicted to encode zinc finger proteins with ∼67% amino acid identity to the protein encoded by the flowering‐time gene CONSTANS (CO). We show that the circadian clock regulates expression of COL1 and COL2 with a peak in transcript levels around dawn. We analyzed transgenic plants misexpressing COL1, COL2 and CO. Unlike CO, altered expression of COL1 and COL2 in transgenic plants had little effect on flowering time. However, analysis of circadian phenotypes in the transgenic plants showed that over‐expression of COL1 can shorten the period of two distinct circadian rhythms. Experiments with the highest COL1 over‐expressing line indicate that its circadian defects are fluence rate‐dependent, suggesting an effect on a light input pathway(s).
The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes
Authors: Ducos Eric , Touzet Pascal , Boutry Marc
Published: March 2003

Cytoplasmic male sterility (CMS) in higher plants has been mainly studied in cultivated species. In most cases, pollen abortion is linked to the presence of an additional mitochondrial polypeptide leading to organelle dysfunction in reproductive tissues. In wild beet, both CMS and hermaphrodite plants coexist in natural populations. The G cytoplasm is widely distributed along the Western European coast, and previous genetic studies have demonstrated that this cytoplasm confers male sterility in beet. In the present study, we have identified two mutations of G mitochondrial genes, each of which results in the production of a respiratory chain complex subunit with an altered molecular weight; the NAD9 subunit has a C‐terminal extension while the COX2 subunit has a truncated C‐terminus. NADH dehydrogenase activity was unchanged in leaves, but cytochrome c oxidase activity was reduced by 50%. Moreover, Western blot analyses revealed that alternative oxidase was more abundant in male sterile G plants than in a fertile control (Nv), suggesting that this alternative pathway might compensate for the cytochrome c oxidase deficiency. Implications of respiratory chain changes and a putative link with CMS are discussed.