bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cymbidium hybrida dihydroflavonol 4‐reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin‐type anthocyanins
Authors: Johnson Eric T. , Yi Hankuil , Shin Byongchul , Oh Boung‐Jun , Cheong Hyeonsook , Choi Giltsu 
Published: March 2003

SummarySome angiosperms are limited to a range of possible flower colors. This limitation can be due to the lack of an anthocyanin biosynthetic gene or to the substrate specificity of a key anthocyanin biosynthetic enzyme, dihydroflavonol 4‐reductase (DFR). Cymbidium hybrida orchid flowers primarily produce cyanidin‐type (pink to red) anthocyanins and lack the pelargonidin‐type (orange to brick‐red) anthocyanins. To investigate the underlying molecular mechanism of this flower color range, we cloned a Cymbidium DFR gene and transformed it into a DFR– petunia line. We found that the Cymbidium DFR did not efficiently reduce dihydrokaempferol (DHK), which is an essential step for pelargonidin production. Phylogenetic analysis of a number of DFR sequences indicate that the inability to catalyze DHK reduction has occurred at least twice during angiosperm evolution. Our results indicate that developing a pelargonidin‐type orange flower color in Cymbidium may require the transformation of a DFR gene that can efficiently catalyze DHK reduction.
MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response?
Authors: Cazalé Anne‐Claire , Droillard Marie‐Jo , Wilson Cathal , Heberle‐Bors Erwin , Barbier‐Brygoo Hélène , Laurière Christiane 
Published: March 2003

SummaryHypoosmotic stress activates a phosphorylation‐dependent oxidative burst. In‐gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen‐activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4‐like. Apigenin, a MAP kinase and cyclin‐dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst ( Cazaléet al. 1998
; Plant Physiol. 116, 659–669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6‐DMAP. However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst. Two‐dimensional in‐gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.
Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues
Authors: Regan Sharon , Bourquin Veronica , Tuominen Hannele , Sundberg Björn 
Published: March 2003

SummaryAccurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non‐specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high‐resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC::uidA, rolC::iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non‐specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.
Isolation of a novel SUMO protein from tomato that suppresses EIX‐induced cell death
Authors: Hanania Uri , Furman‐Matarasso Noa , Ron Mily , Avni Adi 
Published: March 2003

SummaryChallenging tomato or tobacco varieties with ethylene‐inducing xylanase (EIX) from the fungus Trichoderma viride causes rapid induction of plant defence responses leading to programmed cell death. Using the yeast two‐hybrid system, we isolated a novel protein, tomato small ubiquitin‐related modifier protein (T‐SUMO), which specifically interacts with EIX. T‐SUMO, a cytoplasmic protein, is a member of the ubiquitin‐like protein family. It shows homology to human protein sentrin/SUMO1, which suppresses tumour necrosis factor‐induced cell death. Transgenic plants that express T‐SUMO in the sense orientation suppress EIX induction of ethylene biosynthesis and cell death, while in the antisense orientation they enhance EIX‐induced ethylene biosynthesis. These results indicate that T‐SUMO is involved in mediating the signal generated by EIX that leads to induction of plant defence responses.
An abundant TIP expressed in mature highly vacuolated cells
Authors: Karlsson Maria , Johansson Ingela , Bush Max , McCann Maureen C. , Maurel Christophe , Larsson Christer , Kjellbom Per 
Published: March 2003

SummaryAquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS–PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So‐δTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity‐purified So‐δTIP‐specific peptide antibodies, we investigated the subcellular and tissue distribution of So‐δTIP. So‐δTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between δ‐TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So‐δTIP with that of more distantly related TIPs showed similarities with Arabidopsisγ‐TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So‐δTIP may be induced when the large vacuole is formed.
Defective splicing of the first nad4 intron is associated with lack of several complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant
Authors: Brangeon Judy , Sabar Mohammed , Gutierres Sophie , Combettes Bruno , Bove Jérôme , Gendy Cyrille , Chétrit Philippe , Des Francs‐Small Catherine Colas , Pla Magali , Vedel Fernand , De Paepe Rosine 
Published: March 2003

SummaryIn this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a specific mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the first intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two‐dimensional in organello mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or post‐translational steps in the synthesis of other mitochondrial proteins are affected. All of these defects co‐segregated with the abnormal phenotype in the offspring of a NMS1 × wild‐type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.
Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins
Authors: Zhang Shuqun , Liu Yidong , Klessig Daniel F. 
Published: March 2003

SummaryThree protein kinases of 48, 44 and 40 kDa are activated at different stages in tobacco cells treated with fungal elicitins. Previously we demonstrated that the rapidly activated 48 kDa protein kinase is encoded by SIPK. Here we report that the elicitin‐activated 44 kDa kinase is encoded by WIPK. Activation of this kinase occurred 2–4 h after elicitin treatment and was preceded by dramatic increases in WIPK mRNA and protein levels. Studies using actinomycin D and cycloheximide demonstrated that de novo transcription and translation were required for this activation of the kinase activity. Strikingly, the kinetics of WIPK activation following elicitin treatment correlated with the onset of hypersensitive response (HR)‐like cell death. Moreover, staurosporine and K‐252a, two Ser/Thr protein kinase inhibitors that blocked WIPK activation, suppressed cell death. The timing for elicitin‐treated cells to commit to a death program correlated with the appearance of high levels of WIPK activity. These correlative data suggest that WIPK may play a role during HR development in tobacco. Interestingly, a fungal cell‐wall elicitor that does not cause cell death induced WIPK mRNA and protein to similar levels as those observed with the elicitins. However, no corresponding increase in WIPK activity was detected. Thus WIPK appears to be controlled at multiple levels.Keywords: MAP kinases, WIPK, SIPK, elicitin, transcription, translation.
The DNA‐binding activity of Gal4 is inhibited by methylation of the Gal4 binding site in plant chromatin
Authors: Gälweiler Leo , Conlan R. Steven , Mader Patricia , Palme Klaus , Moore Ian 
Published: March 2003

SummaryDerivatives of the Saccharomyces cerevisiae transcription factor Gal4 which act as effective transcription activators in yeast, Drosophila, mammalian cells and plant protoplasts are shown to direct expression from a GUS reporter construct when expressed in transgenic tobacco. However, in comparison to 35S‐GUS controls, Gal4‐mediated expression of the reporter gene was relatively weak and extremely variable. GUS expression was lost as plants matured and it was almost undetectable in most of their progeny. Gal4‐mediated gene expression could be restored by treating tissues with 5‐aza‐cytidine, implicating cytosine methylation in the loss of Gal4‐mediated expression. Restoration of reporter expression was not accompanied by an increase in steady‐state levels of the activator transcript. We propose that the DNA‐binding activity of Gal4 is sensitive to methylation of its binding site in plant chromatin. The Gal4‐DNA co‐crystal predicts that 5‐methylcytosine at either of the outer two positions of the binding site will effectively prevent Gal4 binding. We show that these positions become extensively methylated in transgenic plants and that methylation of Gal4‐binding sites interferes with Gal4 binding in vitro. These observations suggest that the Gal4 DNA‐binding domain is intrinsically sensitive to cytosine methylation and that, despite the success of Gal4‐based expression systems in yeast and Drosophila, Gal4 is not ideal for use in plant gene expression technology.
The characterization of differential calcium signalling in tobacco guard cells
Authors: Wood Nicola T. , Allan Andrew C. , Haley Ann , Viry‐Moussaïd Martine , Trewavas Anthony J. 
Published: March 2003

SummaryTwo novel approaches for the study of Ca2+‐mediated signal transduction in stomatal guard cells are described. Stimulus‐induced changes in guard‐cell cytosolic Ca2+ ([Ca2+]cyt) were monitored using viable stomata in epidermal strips of a transgenic line of Nicotiana plumbaginifolia expressing aequorin (the proteinous luminescent reporter of Ca2+) and in a new transgenic line in which aequorin expression was targeted specifically to the guard cells. The results indicated that abscisic acid (ABA)‐induced stomatal closure was accompanied by increases in [Ca2+]cyt in epidermal strips. In addition to ABA, mechanical and low‐temperature signals directly affected stomatal behaviour, promoting rapid closure. Elevations of guard‐cell [Ca2+]cyt play a key role in the transduction of all three stimuli. However, there were striking differences in the magnitude and kinetics of the three responses. Studies using Ca2+ channel blockers and the Ca2+ chelator EGTA further suggested that mechanical and ABA signals primarily mobilize Ca2+ from intracellular store(s), whereas the influx of extracellular Ca2+ is a key component in the transduction of low‐temperature signals. These results illustrate an aspect of Ca2+ signalling whereby the specificity of the response is encoded by different spatial or kinetic Ca2+ elevations.
Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me‐JA but is dependent on MAPKK activity
Authors: Grant John J. , Yun Byung‐Wook , Loake Gary J. 
Published: March 2003

SummaryRecognition of avirulent microbial pathogens activates an oxidative burst leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. A novel transgenic Arabidopsis line containing a gst1::luc transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of Pseudomonas syringae pv. tomato (Pst). Strong engagement of the oxidative burst was dependent on the presence of functional Pst hrpS and hrpA gene products. Experiments employing pharmacological agents suggested that at least two distinct sources, including an NADPH oxidase and a peroxidase‐type enzyme, contributed to the generation of redox cues. The analysis of gst1 and pal1 gene expression in nahG, coi1 and etr1 plants suggested that engagement of the oxidative burst and cognate redox signalling functioned independently of salicylic acid, methyl jasmonate and ethylene. In contrast, studies using a panel of protein kinase and phosphatase inhibitors and in‐gel kinase assays in these mutant backgrounds suggested that a 48 kDa mitogen‐activated protein kinase (MAPK) activity was required for the activation of gst1 and pal1 in response to redox cues. Thus the engagement of a bifurcating redox signalling pathway possessing a MAPK module may contribute both to the establishment of plant disease resistance, and to the development of cellular protectant mechanisms.
Genes determining pathogenicity to pea are clustered on a supernumerary chromosome in the fungal plant pathogen Nectria haematococca
Authors: Han Yinong , Liu Xiaoguang , Benny Ulla , Kistler H. Corby , VanEtten Hans D. 
Published: March 2003

SummaryThree genes that contribute to the ability of the fungus Nectria haematococca to cause disease on pea plants have been identified. These pea pathogenicity (PEP) genes are within 25 kb of each other and are located on a supernumerary chromosome. Altogether, the PEP gene cluster contains six transcriptional units that are expressed during infection of pea tissue. The biochemical function of only one of the genes is known with certainty. This gene, PDA1, encodes a specific cytochrome P450 that confers resistance to pisatin, an antibiotic produced by pea plants. The three new PEP genes, in addition to PDA1, can independently increase the ability of the fungus to cause lesions on pea when added to an isolate lacking the supernumerary chromosome. Based on predicted amino acid sequences, functions for two of these three genes are hypothesized. The deduced amino acid sequence of another transcribed portion of the PEP cluster, as well as four other open reading frames in the cluster, have a high degree of similarity to known fungal transposases. Several of the features of the PEP cluster – a cluster of pathogenicity genes, the presence of transposable elements, and differences in codon usage and GC content from other portions of the genome – are shared by pathogenicity islands in pathogenic bacteria of plants and animals.
Members of the tomato LeEIL (EIN3‐like) gene family are functionally redundant and regulate ethylene responses throughout plant development
Authors: Tieman Denise M. , Ciardi Joseph A. , Taylor Mark G. , Klee Harry J. 
Published: March 2003

SummaryThe plant hormone ethylene regulates many aspects of growth, development and responses to the environment. The Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) protein is a nuclear‐localized component of the ethylene signal‐transduction pathway with DNA‐binding activity. Loss‐of‐function mutations in this protein result in ethylene insensitivity in Arabidopsis. To gain a better understanding of the ethylene signal‐transduction pathway in tomato, we have identified three homologs of the Arabidopsis EIN3 gene (LeEILs). Each of these genes complemented the ein3‐1 mutation in transgenic Arabidopsis, indicating that all are involved in ethylene signal transduction. Transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene response; reduced expression of multiple tomato LeEIL genes was necessary to reduce ethylene sensitivity significantly. Reduced LeEIL expression affected all ethylene responses examined, including leaf epinasty, flower abscission, flower senescence and fruit ripening. Our results indicate that the LeEILs are functionally redundant and positive regulators of multiple ethylene responses throughout plant development.
The S receptor kinase gene determines dominance relationships in stigma expression of self‐incompatibility in Brassica
Authors: Hatakeyama Katsunori , Takasaki Takeshi , Suzuki Go , Nishio Takeshi , Watanabe Masao , Isogai Akira , Hinata Kokichi 
Published: March 2003

SummarySelf‐incompatibility (SI) in Brassica is sporophytically controlled by the multi‐allelic S locus. SI phenotypes of the stigma and pollen in an S heterozygote are determined by the two S haplotypes it carries; the two haplotypes may be co‐dominant or exhibit a dominant/recessive relationship. Because the S receptor kinase (SRK) gene of the S locus was recently shown to determine the S haplotype specificity of the stigma, we wished to investigate whether SRK also plays a role in the dominance relationships between S haplotypes. We crossed plants carrying an SRK28 transgene with plants homozygous for one of five S haplotypes that are either co‐dominant with, or recessive to, S28 haplotype in the stigma, and analyzed the SI phenotypes of the progeny. In all cases, the SI phenotype of the stigma of plants carrying the SRK28 transgene could be predicted by the known dominance relationships between the S haplotype(s) and the S28 haplotype. Moreover, in the S43 homozygote carrying the SRK28 transgene where the S43 phenotype in the stigma was masked by the presence of the SRK28, the transcript level of SRK28 was found to be much lower than that of SRK43. All these results suggest that the dominance relationships between S haplotypes in the stigma are determined by SRK, but not by virtue of its relative expression level.
Unexpected roles for cryptochrome 2 and phototropin revealed by high‐resolution analysis of blue light‐mediated hypocotyl growth inhibition
Authors: Folta Kevin M. , Spalding Edgar P. 
Published: March 2003

SummaryBlue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de‐etiolation, the transformation of a dark‐grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high‐resolution studies of hypocotyl growth accomplished by computer‐assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the first beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin‐dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin‐dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non‐redundantly to anion‐channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL‐activation of phototropin influences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL‐induced stomatal opening, do not affect any aspect of the growth inhibition within the first 120 min examined here, and NPQ1 does not affect the activation of anion channels.
The auxin signal for protoplast swelling is perceived by extracellular ABP1
Authors: Steffens Bianka , Feckler Christian , Palme Klaus , Christian May , Böttger Michael , Lüthen Hartwig 
Published: March 2003

SummaryProtoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin‐binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C‐terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin‐binding domain, induced a swelling response similar to that caused by auxin treatment. Synthetic C‐terminal oligopeptides of ZmERabp1 also caused a swelling response. These effects occurred regardless of whether the experiments were carried out with homologous (anti‐AtERabp1 antibodies on Arabidopsis protoplasts or anti‐ZmERabp1 antibodies in maize protoplasts) or heterologous immunological tools. The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.