bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ethylene‐inducible PK12 kinase mediates the phosphorylation of SR splicing factors
Authors: Savaldi‐Goldstein Sigal , Sessa Guido , Fluhr Robert 
Published: March 2003

SummaryThe tobacco PK12 is induced by the plant hormone ethylene and is a member of the LAMMER family of protein kinases. Members of this family contain in their C‐terminus a unique ‘EHLAMMERI/VLGPLP’ motif of unknown function, and are related to cyclin‐ and mitogen‐activated protein (MAP)‐dependent kinases. The animal members of this class play a role in differentiation. They phosphorylate and physically interact with serine/arginine‐rich (SR) splicing factors in vivo to alter their activity and the splicing of target mRNAs. SR proteins have been recently described in plants. The capability of PK12 LAMMER kinase to bind and phosphorylate SR proteins was tested in vitro by kinase and binding assays. The tobacco PK12 phosphorylated both animal and plant SR proteins and specifically interacted with the plant splicing factor atSRp34/SR1. In addition, by site‐directed mutagenesis, the LAMMER motif was found to be required for PK12 kinase activity but was not necessary for substrate binding. Consistent with a role in phosphorylation of splicing factors, PK12 was found to localize to the nucleus when transiently over‐expressed in suspension cells.
AtBI‐1, a plant homologue of Bax Inhibitor‐1, suppresses Bax‐induced cell death in yeast and is rapidly upregulated during wounding and pathogen challenge
Authors: Sanchez Pedro , De Torres Zabala Marta , Grant Murray 
Published: March 2003

SummaryExtensive searches have so far failed to identify functional plant homologues of the mammalian apoptotic machinery. Here we report the isolation and characterisation of an Arabidopsis thaliana homologue of human Bax Inhibitor‐1, AtBI‐1, isolated during a differential screen of plants challenged with the phytopathogen Pseudomonas syringae. AtBI is a member of a small gene family in Arabidopsis, members of which display extensive amino acid identity to human BI‐1. AtBI‐1 is also functionally similar to BI‐1 in its ability to suppress the lethal phenotype in yeast conferred by expression of the mammalian proapoptotic protein, Bax. Expression of AtBI‐1 is rapidly upregulated in plants during wounding or pathogen challenge, suggesting a role in responses to biotic and abiotic stress. AtBI‐1 upregulation appears R gene independent and is not markedly affected by mutations required for specific classes of R genes. However, the accumulation of AtBI‐1 message is significantly reduced in coi1, in which defence responses to insects, pathogens and wounding are compromised.
A xylem‐specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress
Authors: Wu Luguang , Joshi Chandrashekhar P. , Chiang Vincent L. 
Published: March 2003

SummaryAngiosperm trees accumulate an elevated amount of highly crystalline cellulose with a concomitant decrease in lignin in the cell walls of tension‐stressed tissues. To investigate the molecular basis of this tree stress response, we cloned a full‐length cellulose synthase (PtCesA) cDNA from developing xylem of aspen (Populus tremuloides). About 90% sequence similarity was found between the predicted PtCesA and cotton GhCesA proteins. Northern blot and in situ hybridization analyses of PtCesA gene transcripts in various aspen tissues, and PtCesA gene promoter‐β‐glucuronidase (GUS) fusion analysis in transgenic tobacco, demonstrated conclusively that PtCesA expression is confined to developing xylem cells during normal plant growth. During mechanical stress induced by stem bending, GUS expression remained in xylem and was induced in developing phloem fibers undergoing tension stress, but was turned off in tissues undergoing compression on the opposite side of the bend. Our results suggest a unique role for PtCesA in cellulose biosynthesis in both tension‐stressed and normal tissues in aspen, and that the on/off control of PtCesA expression may be a part of a signaling mechanism triggering a stress‐related compensatory deposition of cellulose and lignin that is crucial to growth and development in trees.
In vivo targeting of a sunflower oil body protein in yeast secretory (sec) mutants
Authors: Beaudoin Frédéric , Wilkinson Barrie M. , Stirling Colin J. , Napier Johnathan A. 
Published: March 2003

SummaryA sunflower oleosin was expressed in yeast to study the in vivo insertion of the protein into the endoplasmic reticulum (ER) and subsequent transfer to lipid bodies. The oleosin cDNA was expressed in a range of yeast secretory (sec) mutants to determine the precise targeting pathway of the oleosin to the ER. Subcellular fractionation experiments indicated that the signal recognition particle (SRP) is required for oleosin targeting to the ER and hence subsequent deposition on the lipid bodies in vivo. The expression of oleosin in a range of sec61 mutant alleles confirmed the role of the SEC61 translocon in insertion of oleosin into the ER membrane, as well as indicating an unusual substrate/translocon interaction for one particular allele (sec61‐3). Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as determined by pulse–chase analysis. These data therefore provide the first in vivo evidence for the SRP‐dependent targeting of the oleosin to the ER, and the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.
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

SummaryIn 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

SummaryEarly 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

SummaryStudies 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

SummaryScreening 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

SummaryDNA 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

SummaryMcCDPK1 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

SummaryIt 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

SummaryHeat 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

SummaryUrate, 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

SummaryTo 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

SummaryA 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.