bluuebunny/crossref_metadata_2025 huggingface dataset:
dull: rice mutants with tissue‐specific effects on the splicing of the waxy pre‐mRNA
Authors: Isshiki Masayuki , Nakajima Midori , Satoh Hikaru , Shimamoto Ko
Published: March 2003

In the endosperm of japonica rice, du‐1 and du‐2 mutations cause the reduction of amylose contents. It was previously shown that the Wxb allele of rice, which is predominantly distributed in japonica rice, has a mutation in the 5′ splice site of intron 1 resulting in the creation of two weak 5′ splice sites within exon 1. In du‐1 and du‐2 mutants, spliced Wxb transcripts were highly reduced, whereas the processing of transcripts derived from three other genes highly expressed in endosperm was not apparently influenced. Results of competitive RT–PCR analysis indicate that transcripts spliced at the two newly created 5′ splice sites were equally affected in these two mutants. Genetic and molecular analyses of the effects of du‐1 and du‐2 on Wxa pre‐mRNA with normal splice sites indicate that these two mutations do not affect the processing of Wxa pre‐mRNA after splicing, suggesting that du‐1 and du‐2 are mutations of genes required for the efficient splicing of mutated Wxb pre‐mRNA. Furthermore, du‐1 and du‐2 showed differential effects in endosperm and pollen. Although both mutations caused similar effects on the splicing of Wxb transcripts in endosperm, du‐1 caused higher reduction of Wxb mRNA in pollen than in endosperm, while du‐2 had a lesser effect in pollen than in endosperm. Based on these results, we propose that the du‐1 and du‐2 loci of rice encode tissue‐specifically regulated splicing factors that are involved in alternative splicing of pre‐mRNA in rice.
The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death
Authors: Grant Murray , Brown Ian , Adams Sally , Knight Marc , Ainslie Alastair , Mansfield John
Published: March 2003

Early events occurring during the hypersensitive resistance response (HR) were examined using the avrRpm1/RPM1 gene‐for‐gene interaction in Arabidopsis challenged by Pseudomonas syringae pv. tomato. Increases in cytosolic Ca2+ were measured in whole leaves using aequorin‐mediated bioluminescence. During the HR a sustained increase in Ca2+ was observed which was dependent on the presence of both a functional RPM1 gene product and delivery of the cognate avirulence gene product AvrRpm1. The sequence‐unrelated avirulence gene avrB, which also interacts with RPM1, generated a significantly later but similarly prolonged increase in cytosolic Ca2+. Accumulation of H2O2 at reaction sites, as revealed by electron microscopy, occurred within the same time frame as the changes in cytosolic Ca2+. The NADPH oxidase inhibitor diphenylene iodonium chloride did not affect the calcium signature, but did block H2O2 accumulation and the HR. By contrast, the calcium‐channel blocker LaCl3 suppressed the increase in cytosolic Ca2+ as well as H2O2 accumulation and the HR, placing calcium elevation upstream of the oxidative burst.
Evidence for DNA fragmentation triggered in the self‐incompatibility response in pollen of Papaver rhoeas
Authors: Jordan Nicholas D. , Franklin F. Christopher H. , Franklin‐Tong Vernonica E.
Published: March 2003

Studies of the molecular and biochemical basis of self‐incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S‐specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.
The Arabidopsis AtEPR1 extensin‐like gene is specifically expressed in endosperm during seed germination
Authors: Dubreucq Bertrand , Berger Nathalie , Vincent Emmanuel , Boisson Murielle , Pelletier Georges , Caboche Michel , Lepiniec Loïc
Published: March 2003

Screening of 10 000 Arabidopsis transgenic lines carrying a gene‐trap (GUS) construct has been undertaken to identify markers of seed germination. One of these lines showed GUS activity restricted to the endosperm, at the micropylar end of the germinating seed. The genomic DNA flanking the T‐DNA insert was cloned by walking PCR and the insertion was shown to be located 70 bp upstream of a 2285 bp open reading frame (AtEPR1) sharing strong similarities with extensins. The AtEPR1 open reading frame consists of 40 proline‐rich repeats and is expressed in both wild‐type and mutant lines. The expression of the AtEPR1 gene appears to be under positive control of gibberellic acid, but is not downregulated by abscisic acid during seed germination. No expression was detected in organs other than endosperm during seed germination. The putative role of AtEPR1 is discussed in the light of its specific expression in relation to seed germination.
A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity
Authors: Pham Xuan Hoi , Reddy Malireddy Kodandarami , Ehtesham Nasreen Z. , Matta Bharati , Tuteja Narendra
Published: March 2003

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex‐unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino‐acid sequence has eight conserved helicase motifs of the DEAD‐box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP‐dependent DNA and RNA helicase, DNA‐dependent ATPase, and ATP‐binding activities. The protein sequence contains striking homology with eIF‐4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3′ to 5′ direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.
A stress‐induced calcium‐dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two‐component pseudo‐response regulator
Authors: Patharkar O. Rahul , Cushman John C.
Published: March 2003

McCDPK1 is a salinity‐ and drought‐induced calcium‐dependent protein kinase (CDPK) isolated from the common ice plant, Mesembryanthemum crystallinum. A yeast two‐hybrid experiment was performed, using full‐length McCDPK1 and truncated forms of McCDPK1 as baits, to identify interacting proteins. A catalytically impaired bait isolated a cDNA clone encoding a novel protein, CDPK substrate protein 1 (CSP1). CSP1 interacted with McCDPK1 in a substrate‐like fashion in both yeast two‐hybrid assays and wheat germ interaction assays. Furthermore, McCDPK1 was capable of phosphorylating CSP1 in vitro in a calcium‐dependent manner. Our results demonstrate that the use of catalytically impaired and unregulated CDPKs with the yeast two‐hybrid system can accelerate the discovery of CDPK substrates. The deduced CSP1 amino acid sequence indicated that it is a novel member of a class of pseudo‐response regulator‐like proteins that have a highly conserved helix–loop–helix DNA binding domain and a C‐terminal activation domain. McCDPK1 and CSP1 co‐localized to nuclei of NaCl‐stressed ice plants. Csp1 transcript accumulation was not regulated by NaCl or dehydration stress. Our results strongly suggest that McCDPK1 may regulate the function of CSP1 by reversible phosphorylation.
Chain‐length specificities of maize starch synthase I enzyme: studies of glucan affinity and catalytic properties
Authors: Commuri Padmavathi D. , Keeling Peter L.
Published: March 2003

It is widely known that some of the starch synthases and starch‐branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain‐length specificities of the enzyme. Using affinity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of specificity in terms of its substrate–enzyme dissociation constant, but has a greatly elevated affinity for increasing chain lengths of α‐1, 4 glucans. Deletion of the N‐terminal arm of SSI did not affect theKdvalue. Further small deletions of either N‐ or C‐terminal domains resulted in a complete loss of any measurable affinity for its substrate, suggesting that the starch‐affinity domain of SSI is not discrete from the catalytic domain. Greater affinity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest affinity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in affinity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several‐fold using these glucans with extended chain lengths as substrates, and most of the label from [14C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high affinity for the longer glucan chains of dp ≈20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B1chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the specificities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.
Rubisco activase: an enzyme with a temperature‐dependent dual function?
Authors: Rokka Anne , Zhang Lixin , Aro Eva‐Mari
Published: March 2003

Heat treatment of intact spinach leaves was found to induce a unique thylakoid membrane association of an approximately 40 kDa stromal protein. This protein was identified as rubisco activase. Most of the rubisco activase was sequestered to the thylakoid membrane, particularly to the stroma‐exposed regions, during the first 10 min of heat treatment at 42°C. At lower temperatures (38–40°C) the association of rubisco activase with the thylakoid membrane occurred more slowly. The temperature‐dependent association of rubisco activase with the thylakoid membrane was due to a conformational change in the rubisco activase itself, not to heat‐induced alterations in the thylakoid membrane. Association of the 41 kDa isoform of rubisco activase occurred first, followed by the binding of the 45 kDa isoform to the thylakoid membrane. Fractionation of thylakoid membranes revealed a specific association of rubisco activase with thylakoid‐bound polysomes. Our results suggest a temperature‐dependent dual function for rubisco activase. At optimal temperatures it functions in releasing inhibitory sugar phosphates from the active site of Rubisco. During a sudden and unexpected exposure of plants to heat stress, rubisco activase is likely to manifest a second role as a chaperone in association with thylakoid‐bound ribosomes, possibly protecting, as a first aid, the thylakoid associated protein synthesis machinery against heat inactivation.
Effects of urate, a natural inhibitor of peroxynitrite‐mediated toxicity, in the response of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae
Authors: Alamillo Josefa M. , García‐Olmedo Francisco
Published: March 2003

Urate, a natural peroxynitrite scavenger, has been used to investigate the possible role of peroxynitrite during plant‐pathogen interactions. Urate greatly reduced lesion formation in Arabidopsis leaves treated with an abiotic peroxynitrite‐generating system or with a peroxynitrite solution, indicating that it can act as an effective scavenger in planta. In the interaction with the avirulent Pseudomonas syringae pv. phaseolicola (avrRPM1+), cell death in the inoculated area was strongly reduced by urate, without compromising disease resistance. In contrast, urate promoted discrete cell death in response to an isogenic Pseudomonas syringae (avrRPM1–), which did not trigger an HR when inoculated alone, and it induced resistance and arrest of pathogen growth. Scavenging of peroxynitrite did not modify the response of Arabidopsis to an avirulent strain of Xanthomonas campestris pv campestris, that showed a high resistance to NO and peroxynitrite. Our data indicate that peroxynitrite plays a significant role in the responses of plants to Pseudomonas syringae.
Novel marker genes for early leaf development indicate spatial regulation of carbohydrate metabolism within the apical meristem
Authors: Pien Stéphane , Wyrzykowska Joanna , Fleming Andrew J.
Published: March 2003

To identify genes expressed at the earliest stages of leaf development, we have performed a differential display analysis using portions of meristems destined to form leaves. Our analysis led to the identification of five genes showing an asymmetric pattern of gene expression within the meristem associated with leaf formation. Surprisingly, three of these genes encoded enzymes involved in carbohydrate metabolism (ADPglucose pyrophosphorylase, sucrose synthase and an SNF1‐like kinase). Furthermore, specific transcript patterns were responsive to specific sugar and hormonal treatments. The other two genes identified encoded a Phantastica‐like myb transcription factor (associated with the acquisition of leaf dorsiventrality) and CYP85 (a cytochrome P450, which plays a pivotal role in brassinolide metabolism). These data, firstly, identify a novel set of marker genes for the analysis of the earliest stages of leaf formation. Secondly, the function of the proteins encoded by these genes and their expression patterns within the meristem indicate that carbohydrate metabolism is spatially regulated within a tissue involved in key developmental processes. Finally, our data provide the first indication of an asymmetry in gene expression related to hormone biosynthesis in the meristem.
Maize endosperm secretes a novel antifungal protein into adjacent maternal tissue
Authors: Serna Antonio , Maitz Monika , O'Connell Timothy , Santandrea Geraldina , Thevissen Karin , Tienens Koenraad , Hueros Gregorio , Faleri Claudia , Cai Giampiero , Lottspeich Friedrich , Thompson Richard D.
Published: March 2003

A series of endosperm transfer layer‐specific transcripts has been identified in maize by differential screening of a cDNA library of transcripts at 10 days after pollination. Sequence comparisons revealed among this class of cDNAs a novel, small gene family of highly diverged sequences encoding basal layer antifungal proteins (BAPs). The bap genes mapped to two loci on chromosomes 4 and 10. So far, bap‐homologous sequences have been detected only in maize, teosinte and sorghum, and are not present in grasses outside the Andropogoneae tribe. BAP2 is synthesized as a pre‐proprotein, and is processed by successive removal of a signal peptide and a 29‐residue prodomain. The proprotein can be detected exclusively in microsomal membrane‐containing fractions of kernel extracts. Immunolocalization reveals BAP2 to be predominantly located in the placentochalazal cells of the pedicel, adjacent to the basal endosperm transfer layer (BETL) cells, although the BAP2 transcript is found only in the BETL cells. The biological roles of BAP2 propeptide and mature peptide have been investigated by heterologous expression of the proprotein in Escherichia coli, and by tests of its fungistatic activity and that of the fully processed form in vitro. The mature BAP2 peptide exhibits potent broad‐range activity against a range of filamentous fungi, including several plant pathogens.
A highly specific pathogen‐responsive promoter element from the immediate‐early activated CMPG1 gene in Petroselinum crispum
Authors: Kirsch Christoph , Logemann Elke , Lippok Bernadette , Schmelzer Elmon , Hahlbrock Klaus
Published: March 2003

Within the complex signalling network from pathogen‐derived elicitor perception to defense‐related gene activation, some immediate‐early responding genes may have pivotal roles in downstream transcriptional regulation. We have identified the parsley (Petroselinum crispum) ELI17 gene as a particularly fast‐responding gene possessing a new type of W box‐containing, elicitor‐responsive promoter element, E17. Highly selective E17‐mediated reporter gene expression at pathogen infection sites in transgenic Arabidopsis thaliana plants demonstrated the potential of this promoter element for designing new strategies in resistance breeding as well as for further analysis of the early components of defense‐related gene activation mechanisms. The protein encoded by the ELI17 gene exhibits various structural characteristics of established transcription factors and is designated as a CMPG protein according to the first four strictly conserved amino acids defining a newly emerging class of plant‐specific proteins.
Transcriptional activation by the sexual pheromone and wounding: a new gene family from Volvox encoding modular proteins with (hydroxy)proline‐rich and metalloproteinase homology domains
Authors: Hallmann Armin , Amon Peter , Godl Klaus , Heitzer Markus , Sumper Manfred
Published: March 2003

The green alga Volvox represents the simplest kind of multicellular organism: it is composed of only two cell types, somatic and reproductive, making it suitable as a model system. The sexual development of males and females of Volvox carteri is triggered by a sex‐inducing pheromone at a concentration of < 10−16 m. Early biochemical responses to the pheromone involve structural modifications within the extracellular matrix (ECM). By differential screenings of cDNA libraries made from mRNAs of pheromone‐treated Volvox, four novel genes were identified that encode four closely related Volvox metalloproteinases that we use to define a new protein family, the VMPs. The existence of several features common to matrix glycoproteins, such as signal peptides, a (hydroxy)proline content of 12–25%, and Ser(Pro)2−4 repeats, suggest an extracellular localization of the VMPs within the ECM. Synthesis of VMP cDNAs is triggered not only by the sex‐inducing pheromone, but also by wounding, and is restricted to the somatic cell type. Sequence comparisons suggest that the VMPs are members of the MB clan of zinc‐dependent matrix metalloproteinases, although the putative zinc binding site of all VMPs is QEXXHXXGXXH rather than HEXXHXXGXXH. The presence of glutamine instead of histidine in the zinc binding motif suggests a novel family, or even clan, of peptidases. Like the matrixin family of human collagenases, Volvox VMPs exhibit a modular structure: they possess a metalloproteinase homology domain and a (hydroxy)proline‐rich domain, and one of them, VMP4, also has two additional domains. Metalloproteinases seem to be crucial for biochemical modifications of the ECM during development or after wounding in the lower eukaryote Volvox with only two cell types, just as in higher organisms.
The protein kinases AtMAP3Kε1 and BnMAP3Kε1 are functional homologues of S. pombe cdc7p and may be involved in cell division
Authors: Jouannic Stefan , Champion Anthony , Segui‐Simarro José‐Maria , Salimova Ekaterina , Picaud Alain , Tregear James , Testillano Pilar , Risueño Maria‐Carmen , Simanis Viesturs , Kreis Martin , Henry Yves
Published: March 2003

We identified an Arabidopsis thaliana gene, AtMAP3Kε1, and a Brassica napus cDNA, BnMAP3Kε1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7‐related genes in non‐fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT–PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kε1 gene expression is cell cycle‐regulated, with higher expression in G2‐M phases. Our results strongly suggest that the plant cdc7p‐related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.
Inducible high‐level mRNA amplification system by viral replicase in transgenic plants
Authors: Mori Masashi , Fujihara Naoko , Mise Kazuyuki , Furusawa Iwao
Published: March 2003

We have constructed a new system for inducible high‐level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid‐inducible transcription system into the previously developed ‘mRNA amplification system’ where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 × 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid‐inducible promoter. In addition GVG1 × 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S‐driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S‐driven FCP2IFN in the GVG1 × 2FR plant without the treatment and those of the mRNA for IFN were 30–230 times higher than in the previous, non‐inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 × 2FR plant. The advantages and potential uses of this system are also discussed.
Evidence for a role in growth and salt resistance of a plasma membrane H+‐ATPase in the root endodermis
Authors: Vitart Veronique , Baxter Ivan , Doerner Peter , Harper Jeffrey F.
Published: March 2003

The plasma membrane of plant cells is energized by an electrochemical gradient produced by P‐type H+‐ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter‐GUS reporter assays. A disruption of this pump (aha4–1) was identified as a T‐DNA insertion in the middle of the gene (after VFP574). Truncated aha4–1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild‐type plants. Plants homozygous for aha4–1 (–/–) show a subtle reduction in root and shoot growth compared with wild‐type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mm NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4–5‐fold. Interestingly, the aha4–1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild‐type copies of AHA4. These results are consistent with the hypothesis that aha4–1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.
Mapping genes on an integrated sorghum genetic and physical map using cDNA selection technology
Authors: Childs Kevin L. , Klein Robert R. , Klein Patricia E. , Morishige Daryl T. , Mullet John E.
Published: March 2003

Sorghum is an important target of plant genomics. This cereal has unusual tolerance to adverse environments, a small genome (750 Mbp) relative to most other grasses, a diverse germplasm, and utility for comparative genomics with rice, maize and other grasses. In this study, a modified cDNA selection protocol was developed to aid the discovery and mapping of genes across an integrated genetic and physical map of the sorghum genome. BAC DNA from the sorghum genome map was isolated and covalently bound in arrayed tubes for efficient liquid handling. Amplifiable cDNA sequence tags were isolated by hybridization to individual sorghum BACs, cloned and sequenced. Analysis of a fully sequenced sorghum BAC indicated that about 80% of known or predicted genes were detected in the sequence tags, including multiple tags from different regions of individual genes. Data from cDNA selection using the fully sequenced BAC indicate that the occurrence of mislocated cDNA tags is very low. Analysis of 35 BACs (5.25 Mb) from sorghum linkage group B revealed (and therefore mapped) two sorghum genes and 58 sorghum ESTs. Additionally, 31 cDNA tags that had significant homologies to genes from other species were also isolated. The modified cDNA selection procedure described here will be useful for genome‐wide gene discovery and EST mapping in sorghum, and for comparative genomics of sorghum, rice, maize and other grasses.
The ubiquitin‐specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana
Authors: Doelling Jed H. , Yan Ning , Kurepa Jasmina , Walker Joseph , Vierstra Richard D.
Published: March 2003

The ubiquitin/26S proteasome pathway is a major route for selectively degrading cytoplasmic and nuclear proteins in eukaryotes. In this pathway, chains of ubiquitins become attached to short‐lived proteins, signalling recognition and breakdown of the modified protein by the 26S proteasome. During or following target degradation, the attached multi‐ubiquitin chains are released and subsequently disassembled by ubiquitin‐specific proteases (UBPs) to regenerate free ubiquitin monomers for re‐use. Here, we describe Arabidopsis thaliana UBP14 that may participate in this recycling process. Its amino acid sequence is most similar to yeast UBP14 and its orthologues, human IsoT1‐3 and Dictyostelium UbpA, and it can functionally replace yeast UBP14 in a ubp14Δ mutant. Like its orthologues, AtUBP14 can disassemble multi‐ubiquitin chains linked internally via ε‐amino isopeptide bonds using Lys48 and can process some, but not all, translational fusions of ubiquitin linked via α‐amino peptide bonds. However, unlike its yeast and Dictyostelium orthologues, AtUBP14 is essential in Arabidopsis. T‐DNA insertion mutations in the single gene that encodes AtUBP14 cause an embryonic lethal phenotype, with the homozygous embryos arresting at the globular stage. The arrested seeds have substantially increased levels of multi‐ubiquitin chains, indicative of a defect in ubiquitin recycling. Taken together, the data demonstrate an essential role for the ubiquitin/26S proteasome pathway in general and for AtUBP14 in particular during early plant development.
N‐terminal domains of plant poly(ADP‐ribose) polymerases define their association with mitotic chromosomes
Authors: Babiychuk Elena , Van Montagu Marc , Kushnir Sergei
Published: March 2003

Poly(ADP‐ribos)ylation is a reversible protein modification that in higher plants is catalyzed by two structurally different poly(ADP‐ribose) polymerases, App and Zap. In vivo imaging of green‐fluorescent protein (GPF) fusions showed that both Zap and App were associated with chromatin through the cell cycle progression. The in vivo behaviour of the App‐GFP protein fusions can be attributed to the activity of two NASA motifs that mediate protein–protein interactions and nucleic acid binding. Expression of Zap deletion variants revealed that both Zn fingers and helix‐turn‐helix domains contributed to the association with chromosomes, whereas the localization in the nucleoplasm was mostly determined by the Zn fingers. The results highlight novel properties of protein sequences found in plant poly(ADP‐ribose) polymerases and suggest important functions for this class of nuclear enzymes in chromosome dynamics.
Linear DNA intermediates of the Tto1 retrotransposon in Gag particles accumulated in stressed tobacco and Arabidopsis thaliana
Authors: Takeda Shin , Sugimoto Kazuhiko , Kakutani Tetsuji , Hirochika Hirohiko
Published: March 2003

The active transcription of some plant retrotransposons under diverse stress conditions suggests active transposition. However, transposition has been demonstrated only during tissue/cell culture. To examine whether transposition is activated under conditions other than tissue/cell culture, DNA intermediates for retrotransposition of the tobacco retrotransposon Tto1 were analysed. Using transgenic Arabidopsis callus expressing high levels of Tto1 RNA in a ddm1 hypomethylation mutant background, the existence of extrachromosomal Tto1 linear DNA molecules in a Gag‐particle fraction was demonstrated. By combination with ligation‐mediated PCR amplification, we detected Tto1 linear DNA molecules in particle fractions from callus and methyl jasmonate‐treated leaves of tobacco, but not from non‐stressed leaves. Tto1 DNA intermediates could not be detected in the tobacco corolla where Tto1 is expressed. These results indicate that the transcriptional activation of Tto1 by defence‐related stresses leads to the synthesis of DNA intermediates, whereas post‐transcriptional suppression of Tto1 activity is suggested in the corolla.
Low and high affinity amino acid H+‐cotransporters for cellular import of neutral and charged amino acids
Authors: Fischer Wolf‐N. , Loo Donald D. F. , Koch . Wolfgang , Ludewig Uwe , Boorer Kathryn J. , Tegeder Mechthild , Rentsch Doris , Wright Ernest M. , Frommer Wolf B.
Published: March 2003

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) fromArabidopsismediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression inXenopusoocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids.In vivocompetition shows that the capability to transport basic amino acidsin plantamight be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transportedin vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.
Synchronous Arabidopsis suspension cultures for analysis of cell‐cycle gene activity
Authors: Menges Margit , Murray James A.H.
Published: March 2003

Synchronized suspension cultures are powerful tools in plant cell‐cycle studies. However, few Arabidopsis cell cultures are available, and synchrony extending over several sequential phases of the cell cycle has not been reported. Here we describe the first useful synchrony in Arabidopsis, achieved by selecting the rapidly dividing Arabidopsis cell suspensions MM1 and MM2d. Synchrony may be achieved either by removing and re‐supplying sucrose to the growth media or by applying an aphidicolin block/release. Synchronization with aphidicolin produced up to 80% S‐phase cells and up to 92% G2 cells, together with clear separation of different cell‐cycle phases. These synchronization procedures can be used for analysis of gene expression and protein activity. We show that representatives of three CDK gene classes of Arabidopsis (CDKA, CDKB1 and CDKB2) show differential expression timing, and that three CDK inhibitor genes show strikingly different expression patterns during cell‐cycle re‐entry. We propose that ICK2 (KRP2) may have a specific role in this process.
hoc: an Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity
Authors: Catterou Manuella , Dubois Frédéric , Smets Raphael , Vaniet Sébastien , Kichey Thomas , Van Onckelen Harry , Sangwan‐Norreel Brigitte S. , Sangwan Rajbir S.
Published: March 2003

A novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokinin‐overproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth‐regulator mutants. In darkness the hoc mutant is de‐etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two‐ and sevenfold the cytokinin level of wild‐type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the first cytokinin‐overproducing Arabidopsis mutant capable of auto‐regenerating shoots without exogenous growth regulators.
Storekeeper defines a new class of plant‐specific DNA‐binding proteins and is a putative regulator of patatin expression
Authors: Zourelidou Melina , De Torres‐Zabala Marta , Smith Caroline , Bevan Michael W.
Published: March 2003

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber‐specific and sucrose‐inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B‐box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA‐binding protein designated Storekeeper (STK) that specifically recognises the B‐box motif in vitro. Gel shift experiments with an STK‐specific antibody suggest that STK is the B‐box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B‐box motif.
The deposition of suberin lamellae determines the magnitude of cytosolic Ca2+ elevations in root endodermal cells subjected to cooling
Authors: Moore Catherine A. , Bowen Helen C. , Scrase‐Field Sarah , Knight Marc R. , White Philip J.
Published: March 2003

A transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) is thought to be a prerequisite for an appropriate physiological response to both chilling and salt stress. The [Ca2+]cyt is raised by Ca2+ influx to the cytosol from the apoplast and/or intracellular stores. It has been speculated that different signals mobilise Ca2+ from different stores, but little is known about the origin(s) of the Ca2+ entering the cytosol in response to specific environmental challenges. We have utilised the developmentally regulated suberisation of endodermal cells, which is thought to prevent Ca2+ influx from the apoplast, to ascertain whether Ca2+ influx is required to increase [Ca2+]cyt in response to chilling or salt stress. Perturbations in [Ca2+]cyt were studied in transgenic Arabidopsis thaliana, expressing aequorin fused to a modified yellow fluorescent protein solely in root endodermal cells, during slow cooling of plants from 20 to 0.5°C over 5 min and in response to an acute salt stress (0.333 m NaCl). Only in endodermal cells in the apical 4 mm of the Arabidopsis root did [Ca2+]cyt increase significantly during cooling, and the magnitude of the [Ca2+]cyt elevation elicited by cooling was inversely related to the extent of suberisation of the endodermal cell layer. No [Ca2+]cyt elevations were elicited by cooling in suberised endodermal cells. This is consistent with the hypothesis that suberin lamellae isolate the endodermal cell protoplast from the apoplast and, thereby, prevent Ca2+ influx. By contrast, acute salt stress increased [Ca2+]cyt in endodermal cells throughout the root. These results suggest that [Ca2+]cyt elevations, upon slow cooling, depend absolutely on Ca2+ influx across the plasma membrane, but [Ca2+]cyt elevations in response to acute salt stress do not. They also suggest that Ca2+ release from intracellular stores contributes significantly to increasing [Ca2+]cyt upon acute salt stress.
Zinc‐dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria
Authors: Lister Ryan , Mowday Brett , Whelan James , Millar A. Harvey
Published: March 2003

Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin‐sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS‐depleted mitochondria, but did not stimulate import of a non‐carrier control precursor protein, the alternative oxidase. Anion‐exchange separation of IMS components before addition to IMS‐depleted mitochondria revealed a correlation between several 9–10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc‐finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N‐ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.
Phytochrome signalling modulates the SA‐perceptive pathway in Arabidopsis
Authors: Genoud Thierry , Buchala Antony J. , Chua Nam‐Hai , Métraux Jean‐Pierre
Published: March 2003

The interaction of phytochrome signalling with the SA signal transduction pathway has been investigated in Arabidopsis using single and multiple mutants affected in light perception (phyA and phyB deficient) and light‐signal processing (psi2, phytochrome signalling). The induction of PR1 by SA and functional analogues has been found to strictly correlate with the activity of the signalling pathway controlled by both phyA and phyB photoreceptors. In darkness as well as dim light, and independently of a carbohydrate source, SA‐induced PR gene expression as well as the hypersensitive response to pathogens (HR) are strongly reduced. Moreover, the initiation of HR also exhibits a strict dependence upon both the presence and the amplitude of a phytochrome‐elicited signal. The growth of an incompatible strain of bacterial a pathogen (Pseudomonas syringae pv. tomato) was enhanced in phyA‐phyB and decreased in psi2 mutants. While functional chloroplasts were found necessary for the development of an HR, the induction of PRs was strictly dependent on light, but independent of functional chloroplasts. Taken together, these data demonstrate that the light‐induced signalling pathway interacts with the pathogen/SA‐mediated signal transduction route. These results are summarized in a formalism that allows qualitative computer simulation.
CpR18, a novel SAP‐domain plant transcription factor, binds to a promoter region necessary for ABA mediated expression of the CDeT27‐45 gene from the resurrection plant Craterostigma plantagineum Hochst
Authors: Hilbricht Tobias , Salamini Francesco , Bartels Dorothea
Published: March 2003

CDeT27‐45 is a lea‐like gene from the resurrection plant Craterostigma plantagineum (Scrophulariaceae) which is strongly expressed in vegetative tissues in response to dehydration or treatment with abscisic acid (ABA). Expression of the gene is correlated with the acquisition of desiccation tolerance. Nuclear proteins bind to a 29‐bp cis‐regulatory region of the promoter which is essential for transcriptional activation of the CDeT27‐45 gene by ABA. Using a yeast one‐hybrid screen, the cDNA clone CpR18 was isolated, which encodes a protein with specific binding activity for the cis‐regulatory element in the CDeT27‐45 promoter. The protein contains an acidic region, a SAP‐domain, a zinc finger of the C3H‐type, and two motifs which are conserved in proteins from several plant species. One of the conserved regions is rich in basic residues and is predicted to form a helix‐loop‐helix structure. The R18 gene shows high similarities to genomic sequences and ESTs from other plant species. The tissue‐specific expression pattern of the rare R18 mRNA and the distribution of nuclear protein binding activity for the CDeT27‐45 promoter fragment are compared. The R18 protein is indeed localized in the nucleus, and activates transcription of CDeT27‐45 promoter‐GUS fusion constructs in tobacco protoplasts. DNA blot analysis and isolation of genomic clones reveal that two copies of R18 are present in the C. plantagineum genome.
Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells
Authors: Takeuchi Masaki , Ueda Takashi , Yahara Natsuko , Nakano Akihiko
Published: March 2003

Arf GTPases are known to be key regulators of vesicle budding in various steps of membrane traffic in yeast and animal cells. We cloned the Arabidopsis Arf1 homologue, AtArf1, and examined its function. AtArf1 complements yeast arf1 arf2 mutants and its GFP‐fusion is localized to the Golgi apparatus in plant cells like its animal counterpart. The expression of dominant negative mutants of AtArf1 in tobacco and Arabidopsis cultured cells affected the localization of co‐expressed GFP‐tagged proteins in a variety of ways. AtArf1 Q71L and AtArf1 T31N, GTP‐ and GDP‐fixed mutants, respectively, changed the localization of a cis‐Golgi marker, AtErd2‐GFP, from the Golgi apparatus to the endoplasmic reticulum but not that of GFP‐AtRer1B or GFP‐AtSed5. GFP‐AtRer1B and GFP‐AtSed5 were accumulated in aberrant structures of the Golgi by AtArf1 Q71L. A soluble vacuolar protein, sporamin‐GFP, was also located to the ER by AtArf1 Q71L. These results indicate that AtArf1 play roles in the vesicular transport between the ER and the Golgi and in the maintenance of the normal Golgi organization in plant cells.
Five years of Bt cotton in China – the benefits continue
Authors: Pray Carl E. , Huang Jikun , Hu Ruifa , Rozelle Scott
Published: March 2003

Bt cotton is spreading very rapidly in China, in response to demand from farmers for technology that will reduce both the cost of pesticide applications and exposure to pesticides, and will free up time for other tasks. Based on surveys of hundreds of farmers in the Yellow River cotton‐growing region in northern China in 1999, 2000 and 2001, over 4 million smallholders have been able to increase yield per hectare, and reduce pesticide costs, time spent spraying dangerous pesticides, and illnesses due to pesticide poisoning. The expansion of this cost‐saving technology is increasing the supply of cotton and pushing down the price, but prices are still sufficiently high for adopters of Bt cotton to make substantial gains in net income.