bluuebunny/crossref_metadata_2025_split huggingface dataset:
Environmentally sensitive, SA‐dependent defense responses in the cpr22 mutant of Arabidopsis
Authors: Yoshioka Keiko , Kachroo Pradeep , Tsui Frank , Sharma Shashi B. , Shah Jyoti , Klessig Daniel F. 
Published: March 2003

SummaryTo investigate the signaling pathways through which defense responses are activated following pathogen infection, we have isolated and characterized the cpr22 mutant. This plant carries a semidominant, conditional lethal mutation that confers constitutive expression of the pathogenesis‐related (PR) genes PR‐1, PR‐2, PR‐5 and the defensin gene PDF1.2. cpr22 plants also display spontaneous lesion formation, elevated levels of salicylic acid (SA) and heightened resistance to Peronospora parasitica Emco5. The cpr22 locus was mapped to chromosome 2, approximately 2 cm telomeric to the AthB102 marker. By analyzing the progeny of crosses between cpr22 plants and either NahG transgenic plants or npr1 mutants, all of the cpr22‐associated phenotypes except PDF1.2 expression were found to be SA dependent. However, the SA signal transducer NPR1 was required only for constitutive PR‐1 expression. A cross between cpr22 and ndr1–1 mutants revealed that enhanced resistance to P. parasitica is mediated by an NDR1‐dependent pathway, while the other cpr22‐induced defenses are not. Crosses between either coi1–1 or etr1–1 mutants further demonstrated that constitutive PDF1.2 expression is mediated by a JA‐ and ethylene‐dependent pathway. Based on these results, the cpr22 mutation appears to induce its associated phenotypes by activating NPR1‐dependent and NPR1‐independent branches of the SA pathway, as well as an ethylene/JA signaling pathway. Interestingly, the SA‐dependent phenotypes, but not the SA‐independent phenotypes, are suppressed when cpr22 mutants are grown under high humidity.
Retracted: An ∼400 kDa membrane‐associated complex that contains one molecule of the resistance protein Cf‐4
Authors: Rivas Susana , Mucyn Tatiana , Van Den Burg Harrold A. , Vervoort Jacques , Jones Jonathan D. G. 
Published: March 2003

SummaryDespite sharing more than 91% sequence identity, the tomato Cf‐4 and Cf‐9 proteins discriminate between two Cladosporium‐encoded avirulence determinants, Avr4 and Avr9. Comparative studies between Cf‐4 and Cf‐9 are thus of particular interest. To investigate Cf‐4 protein function in initiating defence signalling, we established transgenic tobacco lines and derived cell suspension cultures expressing c‐myc‐tagged Cf‐4. Cf‐4:myc encodes a membrane‐localized glycoprotein of approximately 145 kDa, which confers recognition of Avr4. Elicitation of Cf‐4:myc and Cf‐9:myc tobacco cell cultures with Avr4 and Avr9, respectively, triggered the synthesis of active oxygen species and MAP kinase activation. Additionally, an Agrobacterium‐mediated transient assay was used to express Cf‐4:myc and a newly engineered fusion protein Cf‐4:TAP. Both transiently expressed proteins were found to be functional in an in vivo assay, conferring a hypersensitive response (HR) to Avr4. Consistent with previous observations that Cf‐9 is present in a protein complex, gel filtration analysis of microsomal fractions solubilized with octylglucoside revealed that epitope‐tagged Cf‐4 proteins migrated at a molecular mass of 350–475 kDa. Using blue native gel electrophoresis, the molecular size was confirmed to be approximately 400 kDa. Significantly, this complex appeared to contain only one Cf‐4 molecule, supporting the idea that, as previously described for Cf‐9, additional glycoprotein partners participate with Cf‐4 in the perception of the Avr4 protein. Intriguingly, Cf proteins and Clavata2 (CLV2) of Arabidopsis are highly similar in structure, and the molecular mass of Cf‐4 and CLV complexes is also very similar (400 and 450 kDa, respectively). However, extensive characterization of the Cf‐4 complex revealed essentially identical characteristics to the Cf‐9 complex and significant differences from the CLV2 complex.
Early stages of seed development in Brassica napus: a seed coat‐specific cysteine proteinase associated with programmed cell death of the inner integument
Authors: Wan Lianglu , Xia Qun , Qiu Xiao , Selvaraj Gopalan 
Published: March 2003

SummaryA maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identified a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post‐translational modifications. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows ≈72% identity to BnCysP1. It is, however, leaf senescence‐associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome‐deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post‐fertilization development of this important organ in rapeseed.
Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response
Authors: Yokoi Shuji , Quintero Francisco J. , Cubero Beatriz , Ruiz Maria T. , Bressan Ray A. , Hasegawa Paul M. , Pardo Jose M. 
Published: March 2003

SummaryThe Arabidopsis thaliana vacuolar Na+/H+ antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na+/H+ exchangers led to the identification of five additional AtNHX genes (AtNHX2–6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower‐abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT–PCR. AtNHX1, 2 or 5 suppress, with differential efficacy, the Na+/Li+‐sensitive phenotype of a yeast mutant that is deficient in the endosomal/vacuolar Na+/H+ antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na+ ion compartmentalization and maintain intracellular K+ status. Seedling steady‐state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi‐osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper‐osmotic up‐regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady‐state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2‐1, mutant indicating that the osmotic responsiveness of these genes is ABA‐dependent. An as yet undefined stress signal pathway that is ABA‐ and SOS‐independent apparently controls transcriptional up‐regulation of AtNHX5 expression by hyper‐saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na+.
KCl activates phospholipase D at two different concentration ranges: distinguishing between hyperosmotic stress and membrane depolarization
Authors: Meijer Harold J. G. , Ter Riet Bas , Van Himbergen John A. J. , Musgrave Alan , Munnik Teun 
Published: March 2003

SummaryHyperosmotic stress induces the rapid formation of phosphatidic acid (PA) in Chlamydomonas moewusii via the activation of two signalling pathways: phospholipase D (PLD) and phospholipase C (PLC), the latter in combination with diacylglycerol kinase (DGK) (Munnik et al., 2000). A concomitant increase in cell Ca2+ becomes manifest as deflagellation. When KCl was used as osmoticum we found that two concentration ranges activated deflagellation: one between 50 and 100 mm and another above 200 mm. Deflagellation in low KCl concentrations was complete within 30 sec whereas in high concentrations it took 5 min. PLC was not activated, as it was by high KCl concentrations that cause hyperosmotic stress. Moreover PLD was activated more strongly by low than by high KCl concentrations. Potassium was the most potent monovalent cation based on the induction of deflagellation and the formation of PA and PBut. During treatment, the external medium acidified, indicating an increase in H+‐ATPase activity in order to re‐establish the membrane potential. Activation of PLD and deflagellation at low KCl concentrations were abrogated by treatment with La3+, Gd3+ and EGTA, indicating the dependency on extracellular Ca2+. This suggests that low concentrations of KCl depolarize the plasma membrane, resulting in the activation of H+‐ATPases and opening voltage‐dependent Ca2+± channels, observed as deflagellation and an increase in PLD activity.
Femtosecond near‐infrared laser pulses as a versatile non‐invasive tool for intra‐tissue nanoprocessing in plants without compromising viability
Authors: Tirlapur Uday K. , König Karsten 
Published: March 2003

SummaryIn this report, we describe a highly reproducible femtosecond near‐infrared (NIR) laser‐based nanoprocessing technique that can be used both for non‐invasive intra‐tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra‐precise intra‐tissue nanoprocessing is achieved by the generation of high light intensity (1012 W cm−2) by diffraction‐limited focusing of the radiation of an NIR (λ = 740 and 800 nm) femtosecond titanium‐sapphire laser to a sub‐femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non‐staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non‐invasively within a plant tissue. Further evidence, including two‐photon imaging of chlorophyll fluorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra‐fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio‐temporal control of ontogenetic events and cell–cell interactions, and gravitational biology applications.
In planta protein–protein interactions assessed using a nanovirus‐based replication and expression system
Authors: Aronson Marie N. , Complainville Arnaud , Clérot Danielle , Alcalde Hélène , Katul Lina , Vetten H. Josef , Gronenborn Bruno , Timchenko Tatiana 
Published: March 2003

SummaryThe multipartite genome of the nanovirus Faba bean necrotic yellows virus, which consists of one gene on each DNA component, was exploited to construct a series of virus‐based episomal vectors designed for transient replication and gene expression in plants. This nanovirus based expression system yields high levels of protein which allows isolation of recombinant protein and protein complexes from plant tissues. As examples, we demonstrated in planta interaction between the nanovirus F‐box protein Clink and SKP1, a constituent of the ubiquitin‐dependent protein turnover pathway. Thus, replicative nanovirus vectors provide a simple and efficient means for in planta characterization of protein‐protein interaction.
Light‐signalling pathways leading to the co‐ordinated expression of HEMA1 and Lhcb during chloroplast development in Arabidopsis thaliana
Authors: McCormac Alex C. , Terry Matthew J. 
Published: March 2003

SummaryDuring de‐etiolation, the co‐ordinated synthesis of chlorophyll and the chlorophyll a/b‐binding proteins is critical to the development of functional light‐harvesting complexes. To understand how this co‐ordination is achieved, we have made a detailed study of the light‐regulated signalling pathways mediating the expression of the HEMA1 and Lhcb genes encoding glutamyl‐tRNA reductase, the first committed enzyme of 5‐aminolaevulinic acid formation, and chlorophyll a/b‐binding proteins, respectively. To do this, we have screened 7 photoreceptor and 12 light‐signalling mutants of Arabidopsis thaliana L. for induction of HEMA1 and Lhcb expression in continuous red, far‐red and blue light and following a red pulse. We have categorised these mutants into two groups. The phyA, phyB, phyAphyB, cry1, cry2, cop1, det1, poc1, eid1, and far1 mutations lead to diverse effects on the light regulation of HEMA1, but affect Lhcb expression to a similar degree. The hy1, hy2, hy5, fin219, fhy1, fhy3, spa1, ndpk2, and pat1 mutants also affect light regulation of both HEMA1 and Lhcb expression, but with differences in the relative magnitude of the two responses. The fhy1 and fhy3 mutants show the most significant differences in light regulation between the two genes, with both showing a strong inhibition of HEMA1 expression under continuous red light. These results demonstrate that co‐ordinated regulation of HEMA1 and Lhcb is largely achieved through parallel light regulation mediated by shared phytochrome‐ and cryptochrome‐signalling pathways. However, glutamyl‐tRNA reductase is also required for the synthesis of other tetrapyrroles and this dual role may account for the observed differences in these light‐signalling pathways.
A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula
Authors: Suzuki Hideyuki , Achnine Lahoucine , Xu Ran , Matsuda Seiichi P. T. , Dixon Richard A. 
Published: March 2003

SummaryThe saponins of the model legume Medicago truncatula are glycosides of at least five different triterpene aglycones: soyasapogenol B, soyasapogenol E, medicagenic acid, hederagenin and bayogenin. These aglycones are most likely derived from β‐amyrin, a product of the cyclization of 2,3‐oxidosqualene. Mining M. truncatula EST data sets led to the identification of sequences putatively encoding three early enzymes of triterpene aglycone formation: squalene synthase (SS), squalene epoxidase (SE), and β‐amyrin synthase (β‐AS). SS was functionally characterized by expression in Escherichia coli, two forms of SE by complementation of the yeast erg1 mutant, and β‐AS by expression in yeast. β‐Amyrin was the sole product of the cyclization of squalene epoxide by the recombinant M. truncatulaβ‐AS, as judged by GC–MS and NMR. Transcripts encoding β‐AS, SS and one form of SE were strongly and co‐ordinately induced, associated with accumulation of triterpenes, upon exposure of M. truncatula cell suspension cultures to methyl jasmonate. Sterol composition remained unaffected by jasmonate treatment. Molecular verification of induction of the triterpene pathway in a cell culture system provides a new tool for saponin pathway gene discovery by DNA array‐based approaches.
The chlorate‐resistant and photomorphogenesis‐defective mutant cr88 encodes a chloroplast‐targeted HSP90
Authors: Cao Dongsun , Froehlich John E. , Zhang Hui , Cheng Chi‐Lien 
Published: March 2003

SummaryThe cr88 mutant of Arabidopsis is a novel chlorate‐resistant mutant that displays long hypocotyls in red light, but not in far red or blue light, and is delayed in the greening process. In cotyledons and young leaves, plastids are less developed compared with those of the wild type. In addition, a subset of light‐regulated genes are under‐expressed in this mutant. To understand the pleiotropic phenotypes of cr88, we isolated the CR88 gene through map‐based cloning. We found that CR88 encodes a chloroplast‐targeted 90‐kDa heat shock protein (HSP90). The CR88 gene is expressed at highest levels during early post‐germination stages and in leaves and reproductive organs. It is constitutively expressed but is also light and heat shock inducible. Chloroplast import experiments showed that the protein is localized to the stroma compartment of the chloroplast. The possible function of an HSP90 in the chloroplast and a plausible explanation of the pleiotropic phenotypes observed in cr88 are discussed.
Alteration of GCN5 levels in maize reveals dynamic responses to manipulating histone acetylation
Authors: Bhat Riyaz A. , Riehl Marcus , Santandrea Geraldina , Velasco Riccardo , Slocombe Stephen , Donn Günter , Steinbiss Hans‐Henning , Thompson Richard D. , Becker Heinz‐Albert 
Published: March 2003

SummaryThe role played by histone acetyltransferase (HAT), GCN5, in transcriptional co‐activation has been analysed in detail in yeast and mammals. Here, we present the cloning and expression pattern of Zmgcn5, the maize homologue. The enzymatic activity of the recombinant ZmGCN5 was analysed with histone and nucleosome substrates. In situ hybridisation of developing maize kernels using Zmgcn5 as probe shows that the transcript is concentrated in rapidly dividing cells. To investigate the role of ZmGCN5 in the transcription of specific plant genes, direct protein–protein interactions were tested. A cDNA clone encoding a putative interacting partner in GCN5‐adapter complexes, ZmADA2, was isolated and the interaction between ZmGCN5 and ZmADA2 was confirmed by a GST‐spin down experiment. Co‐immunoprecipitation of the plant transcriptional activator Opaque‐2 and ZmADA2 in nuclear extracts suggests ADA2/GCN5‐containing complexes to mediate transcriptional activation by binding of this bZIP factor. For a more general analysis of the effects of histone acetylation on plant gene expression, 2500 ESTs spotted on filters were hybridised with cDNA probes derived either from maize cell lines treated with Trichostatin A (TSA), or from a transgenic line expressing the ZmGCN5 antisense transcript. Several sequences showing marked changes in abundance were confirmed by RNA blot analysis. Inhibition of histone deacetylation with TSA is accompanied by a decrease in the abundance of ZmGCN5 acetylase protein, but by increases in mRNAs for histones H2A, H2B, H3 and H4. The elevated histone mRNA levels were not reflected in increasing histone protein concentrations, suggesting hyperacetylated histones arising from TSA treatment may be preferentially degraded and substituted by de novo synthesised histones. The ZmGCN5 antisense material showed suppression of the endogenous ZmGCN5 transcript and the profiling analysis revealed increased mRNA levels for H2A, H2B and H4. Furthermore, in the antisense line, a reduction in the amount of the RPD3‐type HD1B‐I histone deacetylase protein was observed. A model for linked regulation of histone acetylation and histone mRNA transcription is discussed.
ADP‐glucose pyrophosphorylase from potato tuber: site‐directed mutagenesis of homologous aspartic acid residues in the small and large subunits
Authors: Frueauf Jeremiah B. , Ballicora Miguel A. , Preiss Jack 
Published: March 2003

SummaryAsp142in the homotetrameric ADP‐glucose pyrophosphorylase (ADP‐Glc PPase) enzyme fromEscherichia coliwas demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001)J. Biol. Chem., 276, 46319–46325]. The residue is highly conserved throughout the family of ADP‐Glc PPases, as well as throughout the super‐family of sugar–nucleotide pyrophosphorylases. In the heterotetrameric ADP‐Glc PPase from potato (Solanum tuberosumL.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP‐Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP‐Glc PPase from potato tuber, we used site‐directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP‐Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160Emutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160Emutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3‐phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145in the small subunit is necessary for enzyme catalysis.
The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor
Authors: Sorensen Anna‐Marie , Kröber Sandra , Unte Ulrike S. , Huijser Peter , Dekker Koen , Saedler Heinz 
Published: March 2003

SummaryVisual screening of a T‐DNA mutagenised population of Arabidopsis thaliana for an absence of silique elongation lead to the isolation of the aborted microspores (ams) mutant that shows a sporophytic recessive male sterile phenotype. Homozygous mutant plants are completely devoid of mature pollen. Pollen degeneration occurs shortly after release of the microspores from the tetrad, prior to pollen mitosis I. Premature tapetum and microspore degeneration are the primary defects caused by this lesion, while a secondary effect is visualised in the stamen filaments, which are reduced in length and lie beneath the receptive stigma at flower opening. The disrupted gene was isolated and revealed a T‐DNA element to be inserted into the eighth exon of a basic helix‐loop‐helix (bHLH) gene located on chromosome II. This protein sequence contains a basic DNA binding domain and two alpha helices separated by a loop, typical of a transcription factor belonging to the MYC sub family of bHLH genes. Therefore, AMS plays a crucial role in tapetal cell development and the post‐meiotic transcriptional regulation of microspore development within the developing anther.