bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant Defensins
Authors: Spelbrink Robert G. , Dilmac Nejmi , Allen Aron , Smith Thomas J. , Shah Dilip M. , Hockerman Gregory H.
Published: August 2004

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized α/β-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca2+. By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31–45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca2+ channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca2+ channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.
Metabolic Discrimination ofCatharanthus roseusLeaves Infected by Phytoplasma Using 1H-NMR Spectroscopy and Multivariate Data Analysis
Authors: Choi Young Hae , Tapias Elisabet Casas , Kim Hye Kyong , Lefeber Alfons W.M. , Erkelens Cornelis , Verhoeven Jacobus Th.J. , Brzin Jernej , Zel Jana , Verpoorte Robert
Published: August 2004

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the 1H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.
Phosphorylation of Transitory Starch Is Increased during Degradation
Authors: Ritte Gerhard , Scharf Anke , Eckermann Nora , Haebel Sophie , Steup Martin
Published: August 2004

The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied 32P orthophosphate into starch. Illuminated cells incorporated 32P into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with 32P/31P orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch.
The Role of the C4 Pathway in Carbon Accumulation and Fixation in a Marine Diatom
Authors: Reinfelder John R. , Milligan Allen J. , Morel François M.M.
Published: August 2004

The role of a C4 pathway in photosynthetic carbon fixation by marine diatoms is presently debated. Previous labeling studies have shown the transfer of photosynthetically fixed carbon through a C4 pathway and recent genomic data provide evidence for the existence of key enzymes involved in C4 metabolism. Nonetheless, the importance of the C4 pathway in photosynthesis has been questioned and this pathway is seen as redundant to the known CO2 concentrating mechanism of diatoms. Here we show that the inhibition of phosphoenolpyruvate carboxylase (PEPCase) by 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate resulted in a more than 90% decrease in whole cell photosynthesis in Thalassiosira weissflogii cells acclimated to low CO2 (10 μ  m), but had little effect on photosynthesis in the C3 marine Chlorophyte, Chlamydomonas sp. In 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate-treated T. weissflogii cells, elevated CO2 (150 μ  m) or low O2 (80–180 μ  m) restored photosynthesis to the control rate linking PEPCase inhibition with CO2 supply in this diatom. In C4 organic carbon-inorganic carbon competition experiments, the 12C-labeled C4 products of PEPCase, oxaloacetic acid and its reduced form malic acid suppressed the fixation of 14C-labeled inorganic carbon by 40% to 50%, but had no effect on O2 evolution in photosynthesizing diatoms. Oxaloacetic acid-dependent O2 evolution in T. weissflogii was twice as high in cells acclimated to 10 μ  m rather than 22 μ  m CO2, indicating that the use of C4 compounds for photosynthesis is regulated over the range of CO2 concentrations observed in marine surface waters. Short-term 14C uptake (silicone oil centrifugation) and CO2 release (membrane inlet mass spectrometry) experiments that employed a protein denaturing cell extraction solution containing the PEPCKase inhibitor mercaptopicolinic acid revealed that much of the carbon taken up by diatoms during photosynthesis is stored as organic carbon before being fixed in the Calvin cycle, as expected if the C4 pathway functions as a CO2 concentrating mechanism. Together these results demonstrate that the C4 pathway is important in carbon accumulation and photosynthetic carbon fixation in diatoms at low (atmospheric) CO2.
The Spatially Variable Inhibition by Water Deficit of Maize Root Growth Correlates with Altered Profiles of Proton Flux and Cell Wall pH
Authors: Fan Ling , Neumann Peter M.
Published: August 2004

Growth of elongating primary roots of maize (Zea mays) seedlings was approximately 50% inhibited after 48 h in aerated nutrient solution under water deficit induced by polyethylene glycol 6000 at −0.5 MPa water potential. Proton flux along the root elongation zone was assayed by high resolution analyses of images of acid diffusion around roots contacted for 5 min with pH indicator gel. Profiles of root segmental elongation correlated qualitatively and quantitatively (r  2 = 0.74) with proton flux along the surface of the elongation zone from water-deficit and control treatments. Proton flux and segmental elongation in roots under water deficit were remarkably well maintained in the region 0 to 3 mm behind the root tip and were inhibited from 3 to 10 mm behind the tip. Associated changes in apoplastic pH inside epidermal cell walls were measured in three defined regions along the root elongation zone by confocal laser scanning microscopy using a ratiometric method. Finally, external acidification of roots was shown to specifically induce a partial reversal of growth inhibition by water deficit in the central region of the elongation zone. These new findings, plus evidence in the literature concerning increases induced by acid pH in wall-extensibility parameters, lead us to propose that the apparently adaptive maintenance of growth 0 to 3 mm behind the tip in maize primary roots under water deficit and the associated inhibition of growth further behind the tip are related to spatially variable changes in proton pumping into expanding cell walls.
Pathogen- and NaCl-Induced Expression of the SCaM-4 Promoter Is Mediated in Part by a GT-1 Box That Interacts with a GT-1-Like Transcription Factor
Authors: Park Hyeong Cheol , Kim Man Lyang , Kang Yun Hwan , Jeon Joo Mi , Yoo Jae Hyuk , Kim Min Chul , Park Chan Young , Jeong Jae Cheol , Moon Byeong Cheol , Lee Ju Huck , Yoon Hae Won , Lee Sung-Ho , Chung Woo Sik , Lim Chae Oh , Lee Sang Yeol , Hong Jong Chan , Cho Moo Je
Published: August 2004

The Ca2+-binding protein calmodulin mediates cellular Ca2+ signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions −858 and −728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.
Types and Frequencies of Sequencing Errors in Methyl-Filtered and High C0t Maize Genome Survey Sequences
Authors: Fu Yan , Hsia An-Ping , Guo Ling , Schnable Patrick S.
Published: August 2004

The Maize Genome Sequencing Consortium has deposited into GenBank more than 850,000 maize (Zea mays) genome survey sequences (GSSs) generated via two gene enrichment strategies, methylation filtration and high-C0t (HC) fractionation. These GSSs are a valuable resource for generating genome assemblies and the discovery of single nucleotide polymorphisms and nearly identical paralogs. Based on the rate of mismatches between 183 GSSs (105 methylation filtration + 78 HC) and 10 control genes, the rate of sequencing errors in these GSSs is 2.3 × 10−3. As expected many of these errors were derived from insufficient vector trimming and base-calling errors. Surprisingly, however, some errors were due to cloning artifacts. These G•C to A•T transitions are restricted to HC clones; over 40% of HC clones contain at least one such artifact. Because it is not possible to distinguish the cloning artifacts from biologically relevant polymorphisms, HC sequences should be used with caution for the discovery of single nucleotide polymorphisms or paramorphisms. The average rate of sequencing errors was reduced 6-fold (to 3.6 × 10−4) by applying more stringent trimming parameters. This trimming resulted in the loss of only 11% of the bases (15,469/144,968). Due to redundancy among GSSs this more stringent trimming reduced coverage of promoters, exons, and introns by only 0%, 1%, and 4%, respectively. Hence, at the cost of a very modest loss of gene coverage, the quality of these maize GSSs can approach Bermuda standards, even prior to assembly.
Biochemical and Structural Characterization of Benzenoid Carboxyl Methyltransferases Involved in Floral Scent Production in Stephanotis floribunda and Nicotiana suaveolens
Authors: Pott Marcella B. , Hippauf Frank , Saschenbrecker Sandra , Chen Feng , Ross Jeannine , Kiefer Ingrid , Slusarenko Alan , Noel Joseph P. , Pichersky Eran , Effmert Uta , Piechulla Birgit
Published: August 2004

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K  m value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K  m values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability.
Proteome Reference Maps of Vegetative Tissues in Pea. An Investigation of Nitrogen Mobilization from Leaves during Seed Filling
Authors: Schiltz Séverine , Gallardo Karine , Huart Myriam , Negroni Luc , Sommerer Nicolas , Burstin Judith
Published: August 2004

A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.
Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily
Authors: Martin Diane M. , Fäldt Jenny , Bohlmann Jörg
Published: August 2004

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (−)-limonene synthase, (−)-α/β-pinene synthase, and (−)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-α-farnesene synthase, and E-α-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.
Gene Expression Profiling of the Tetrapyrrole Metabolic Pathway in Arabidopsis with a Mini-Array System
Authors: Matsumoto Fuminori , Obayashi Takeshi , Sasaki-Sekimoto Yuko , Ohta Hiroyuki , Takamiya Ken-ichiro , Masuda Tatsuru
Published: August 2004

Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.
AtPng1p. The First Plant Transglutaminase
Authors: Della Mea Massimiliano , Caparrós-Ruiz David , Claparols Inmaculada , Serafini-Fracassini Donatella , Rigau Joan
Published: August 2004

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines—spermine (Spm) > spermidine (Spd) > putrescine (Put)—and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the γ-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of γ-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca2+- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.
Functional Characterization of Enzymes Forming Volatile Esters from Strawberry and Banana
Authors: Beekwilder Jules , Alvarez-Huerta Mayte , Neef Evert , Verstappen Francel W.A. , Bouwmeester Harro J. , Aharoni Asaph
Published: August 2004

Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria × ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess the potential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants overexpressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is an important parameter to consider when engineering volatile ester formation in plants.
Roots, Cycles and Leaves. Expression of the Phosphoenolpyruvate Carboxylase Kinase Gene Family in Soybean
Authors: Sullivan Stuart , Jenkins Gareth I. , Nimmo Hugh G.
Published: August 2004

Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism of higher plants. This phosphorylation is controlled largely at the level of expression of PEPc kinase (PPCK) genes. We have analyzed the expression of both PPCK genes and the PEPC genes that encode PEPc in soybean (Glycine max). Soybean contains at least four PPCK genes. We report the genomic and cDNA sequences of these genes and demonstrate the function of the gene products by in vitro expression and enzyme assays. For two of these genes, GmPPCK2 and GmPPCK3, transcript abundance is highest in nodules and is markedly influenced by supply of photosynthate from the shoots. One gene, GmPPCK4, is under robust circadian control in leaves but not in roots. Its transcript abundance peaks in the latter stages of subjective day, and its promoter contains a sequence very similar to the evening element found in Arabidopsis genes expressed at this time. We report the expression patterns of five PEPC genes, including one encoding a bacterial-type PEPc lacking the phosphorylation site of the plant-type PEPcs. The PEPc expression patterns do not match those of any of the PPCK genes, arguing against the existence of specific PEPc-PPCK expression partners. The PEPC and PPCK gene families in soybean are significantly more complex than previously understood.
Herbivore-Induced Defense Response in a Model Legume. Two-Spotted Spider Mites Induce Emission of (E)-β-Ocimene and Transcript Accumulation of (E)-β-Ocimene Synthase in Lotus japonicus
Authors: Arimura Gen-ichiro , Ozawa Rika , Kugimiya Soichi , Takabayashi Junji , Bohlmann Jörg
Published: August 2004

Indirect defense of plants against herbivores often involves the induced emission of volatile infochemicals including terpenoids that attract natural enemies of the herbivores. We report the isolation and characterization of a terpene synthase cDNA (LjEβOS) from a model legume, Lotus japonicus. Recombinant LjEβOS enzyme produced (E)-β-ocimene (98%) and its Z-isomer (2%). Transcripts of LjEβOS were induced in L. japonicus plants infested with two-spotted spider mites (Tetranychus urticae), coinciding with increasing emissions of (E)-β-ocimene as well as other volatiles, (Z)-3-hexenyl acetate and (E)-4,8-dimethyl-1,3,7-nonatriene, by the infested plants. We suggest that LjEβOS is involved in the herbivore-induced indirect defense response of spider mite-infested L. japonicus via de novo formation and emission (E)-β-ocimene. Mechanical wounding of the leaves or application of alamethicin (ALA), a potent fungal elicitor of plant volatile emission, also induced transiently increased levels of LjEβOS transcripts in L. japonicus. However, wounding or ALA did not result in elevated release of (E)-β-ocimene. Differences in volatile emissions after herbivory, mechanical wounding, or treatment with ALA suggest that neither a single mechanical wounding event nor ALA simulate the effect of herbivore activity and indicate that herbivore-induced emission of (E)-β-ocimene in L. japonicus involves control mechanisms in addition to up-regulation of LjEβOS transcripts.
Ovule Abortion in Arabidopsis Triggered by Stress
Authors: Sun Kelian , Hunt Kimberly , Hauser Bernard A.
Published: August 2004

Environmental stresses frequently decrease plant fertility. In Arabidopsis, the effect of salt stress on reproduction was examined using plants grown in hydroponic medium. Salt stress inhibited microsporogenesis and stamen filament elongation. Because plants grown in hydroponic media can be rapidly and transiently stressed, the minimum inductive treatment to cause ovule abortion could be determined. Nearly 90% of the ovules aborted when roots were incubated for 12 h in a hydroponic medium supplemented with 200 mm NaCl. The anatomical effects of salt stress on maternal organs were distinct from those in the gametophyte. A fraction of cells in the chalaza and integuments underwent DNA fragmentation and programmed cell death. While three-fourths of the gametophytes aborted prior to fertilization, DNA fragmentation was not detected in these cells. Those gametophytes that survived were fertilized and formed embryos. However, very few of these developing embryos formed seeds; most senesced during seed development. Thus, during seed formation, there were multiple points where stress could prematurely terminate plant reproduction. These decreases in fecundity are discussed with respect to the hypothesis of serial adjustment of maternal investment.
Transient Release of Oxygenated Volatile Organic Compounds during Light-Dark Transitions in Grey Poplar Leaves
Authors: Graus Martin , Schnitzler Jörg-Peter , Hansel Armin , Cojocariu Cristian , Rennenberg Heinz , Wisthaler Armin , Kreuzwieser Jürgen
Published: August 2004

In this study, we investigated the prompt release of acetaldehyde and other oxygenated volatile organic compounds (VOCs) from leaves of Grey poplar [Populus x canescens (Aiton) Smith] following light-dark transitions. Mass scans utilizing the extremely fast and sensitive proton transfer reaction-mass spectrometry technique revealed the following temporal pattern after light-dark transitions: hexenal was emitted first, followed by acetaldehyde and other C6-VOCs. Under anoxic conditions, acetaldehyde was the only compound released after switching off the light. This clearly indicated that hexenal and other C6-VOCs were released from the lipoxygenase reaction taking place during light-dark transitions under aerobic conditions. Experiments with enzyme inhibitors that artificially increased cytosolic pyruvate demonstrated that the acetaldehyde burst after light-dark transition could not be explained by the recently suggested pyruvate overflow mechanism. The simulation of light fleck situations in the canopy by exposing leaves to alternating light-dark and dark-light transitions or fast changes from high to low photosynthetic photon flux density showed that this process is of minor importance for acetaldehyde emission into the Earth's atmosphere.
A Novel Auxin Conjugate Hydrolase from Wheat with Substrate Specificity for Longer Side-Chain Auxin Amide Conjugates
Authors: Campanella James J. , Olajide Adebanke F. , Magnus Volker , Ludwig-Müller Jutta
Published: August 2004

This study investigates how the ILR1-like indole acetic acid (IAA) amidohydrolase family of genes has functionally evolved in the monocotyledonous species wheat (Triticum aestivum). An ortholog for the Arabidopsis IAR3 auxin amidohydrolase gene has been isolated from wheat (TaIAR3). The TaIAR3 protein hydrolyzes negligible levels of IAA-Ala and no other IAA amino acid conjugates tested, unlike its ortholog IAR3. Instead, TaIAR3 has low specificity for the ester conjugates IAA-Glc and IAA-myoinositol and high specificity for the conjugates of indole-3-butyric acid (IBA-Ala and IBA-Gly) and indole-3-propionic-acid (IPA-Ala) so far tested. TaIAR3 did not convert the methyl esters of the IBA conjugates with Ala and Gly. IBA and IBA conjugates were detected in wheat seedlings by gas chromatography-mass spectrometry, where the conjugate of IBA with Ala may serve as a natural substrate for this enzyme. Endogenous IPA and IPA conjugates were not detected in the seedlings. Additionally, crude protein extracts of wheat seedlings possess auxin amidohydrolase activity. Temporal expression studies of TaIAR3 indicate that the transcript is initially expressed at day 1 after germination. Expression decreases through days 2, 5, 10, 15, and 20. Spatial expression studies found similar levels of expression throughout all wheat tissues examined.
Brassinosteroid Deficiency Due to Truncated Steroid 5α-Reductase Causes Dwarfism in thelkMutant of Pea
Authors: Nomura Takahito , Jager Corinne E. , Kitasaka Yukiko , Takeuchi Keiichi , Fukami Motohiro , Yoneyama Koichi , Matsushita Yasuhiko , Nyunoya Hiroshi , Takatsuto Suguru , Fujioka Shozo , Smith Jennifer J. , Kerckhoffs L. Huub J. , Reid James B. , Yokota Takao
Published: August 2004

The endogenous brassinosteroids in the dwarf mutant lk of pea (Pisum sativum) were quantified by gas chromatography-selected ion monitoring. The levels of castasterone, 6-deoxocastasterone, and 6-deoxotyphasterol in lk shoots were reduced 4-, 70-, and 6-fold, respectively, compared with those of the wild type. The fact that the application of brassinolide restored the growth of the mutant indicated that the dwarf mutant lk is brassinosteroid deficient. Gas chromatography-selected ion monitoring analysis of the endogenous sterols in lk shoots revealed that the levels of campestanol and sitostanol were reduced 160- and 10-fold, respectively, compared with those of wild-type plants. These data, along with metabolic studies, showed that the lk mutant has a defect in the conversion of campest-4-en-3-one to 5α-campestan-3-one, which is a key hydrogenation step in the synthesis of campestanol from campesterol. This defect is the same as that found in the Arabidopsis det2 mutant and the Ipomoea nil kbt mutant. The pea gene homologous to the DET2 gene, PsDET2, was cloned, and it was found that the lk mutation would result in a putative truncated PsDET2 protein. Thus it was concluded that the short stature of the lk mutant is due to a defect in the steroidal 5α-reductase gene. This defect was also observed in the callus induced from the lk mutant. Biosynthetic pathways involved in the conversion of campesterol to campestanol are discussed in detail.
An In Vivo Analysis of the Effect of Season-Long Open-Air Elevation of Ozone to Anticipated 2050 Levels on Photosynthesis in Soybean
Authors: Morgan Patrick B. , Bernacchi Carl J. , Ort Donald R. , Long Stephen P.
Published: August 2004

Rising atmospheric carbon dioxide concentration ([CO2]) is widely recognized, but less appreciated is a concomitant rise in tropospheric ozone concentration ([O3]). In industrialized countries, [O3] has risen by 0.5% to 2.5% per year. Tropospheric [O3] is predicted to reach a global mean of >60 nL L−1 by 2050 with greater averages locally. Previous studies in enclosures suggest that this level of [O3] will decrease leaf photosynthesis, thereby limiting growth and yield of Glycine max L. Merr. SoyFACE (Soybean Free Air gas Concentration Enrichment) is the first facility to elevate atmospheric [O3] (approximately 1.2× current) in replicated plots under completely open-air conditions within an agricultural field. Measurements of gas exchange (assimilation versus light and assimilation versus intercellular [CO2]) were made on excised leaves from control and treatment plots (n = 4). In contrast to expectations from previous chamber studies, elevated [O3] did not alter light-saturated photosynthesis (A  sat, P = 0.09), carboxylation capacity (V  c,max, P = 0.82), or maximum electron transport (J  max, P = 0.66) for the topmost most recently fully expanded leaf at any stage of crop development. Leaves formed during the vegetative growth stage did not show a significant ozone-induced loss of photosynthetic capacity as they aged. Leaves formed during flowering did show a more rapid loss of photosynthetic capacity as they aged in elevated [O3]. A  sat, V  c,max, and J  max (P = 0.04, 0.004, and 0.002, respectively) were decreased 20% to 30% by treatment with ozone. This is noteworthy since these leaves provide photosynthate to the developing grain. In conclusion, a small (approximately 20%) increase in tropospheric [O3] did not significantly alter photosynthetic capacity of newly expanded leaves, but as these leaves aged, losses in photosynthetic carbon assimilation occurred.
Transcriptome Profiling of the Response of Arabidopsis Suspension Culture Cells to Suc Starvation
Authors: Contento Anthony L. , Kim Sang-Jin , Bassham Diane C.
Published: August 2004

Upon encountering nutrient stress conditions, plant cells undergo extensive metabolic changes and induce nutrient recycling pathways for their continued survival. The role of nutrient mobilization in the response of Arabidopsis suspension cells to Suc starvation was examined. Vacuolar autophagy was induced within 24 h of starvation, with increased expression of vacuolar proteases that are likely to be required for degradation of cytoplasmic components delivered to the vacuole, and thus for nutrient recycling. After 48 h of starvation, culture viability began to decrease, and substantial cell death was evident by 72 h. To provide further insight into the pathways required for survival during Suc deficit, transcriptional profiling during Suc starvation was performed using the ATH1 GeneChip array containing 22,810 probe sets. A significant increase in transcript levels was observed for 343 genes within 48 h of starvation, indicating a response to nutrient stress that utilizes the recycling of cellular components and nutrient scavenging for maintaining cell function, the protection of the cell from death through activation of various defense and stress response pathways, and regulation of these processes by specific protein kinases and transcription factors. These physiological and molecular data support a model in which plant cells initiate a coordinated response of nutrient mobilization at the onset of Suc depletion that is able to maintain cell viability for up to 48 h. After this point, genes potentially involved in cell death increase in expression, whereas those functioning in translation and replication decrease, leading to a decrease in culture viability and activation of cell death programs.
Characterization of a Root-Specific Arabidopsis Terpene Synthase Responsible for the Formation of the Volatile Monoterpene 1,8-Cineole
Authors: Chen Feng , Ro Dae-Kyun , Petri Jana , Gershenzon Jonathan , Bohlmann Jörg , Pichersky Eran , Tholl Dorothea
Published: August 2004

Arabidopsis is emerging as a model system to study the biochemistry, biological functions, and evolution of plant terpene secondary metabolism. It was previously shown that the Arabidopsis genome contains over 30 genes potentially encoding terpene synthases (TPSs). Here we report the characterization of a monoterpene synthase encoded by two identical, closely linked genes, At3g25820 and At3g25830. Transcripts of these genes were detected almost exclusively in roots. An At3g25820/At3g25830 cDNA was expressed in Escherichia coli, and the protein thus produced was shown to catalyze the formation of 10 volatile monoterpenes from geranyl diphosphate, with 1,8-cineole predominating. This protein was therefore designated AtTPS-Cin. The purified recombinant AtTPS-Cin displayed similar biochemical properties to other known monoterpene synthases, except for a relatively low K  m value for geranyl diphosphate of 0.2 μ  m. At3g25820/At3g25830 promoter activity, measured with a β-glucuronidase (GUS) reporter gene, was primarily found in the epidermis, cortex, and stele of mature primary and lateral roots, but not in the root meristem or the elongation zone. Although the products of AtTPS-Cin were not detected by direct extraction of plant tissue, the recent report of 1,8-cineole as an Arabidopsis root volatile (Steeghs M, Bais HP, de Gouw J, Goldan P, Kuster W, Northway M, Fall R, Vivanco JM [2004] Plant Physiol 135: 47–58) suggests that the enzyme products may be released into the rhizosphere rather than accumulated. Among Arabidopsis TPSs, AtTPS-Cin is most similar to the TPS encoded by At3g25810, a closely linked gene previously shown to be exclusively expressed in flowers. At3g25810 TPS catalyzes the formation of a set of monoterpenes that is very similar to those produced by AtTPS-Cin, but its major products are myrcene and (E)-β-ocimene, and it does not form 1,8-cineole. These data demonstrate that divergence of organ expression pattern and product specificity are ongoing processes within the Arabidopsis TPS family.
Characterization of the Complex Locus of Bean Encoding Polygalacturonase-Inhibiting Proteins Reveals Subfunctionalization for Defense against Fungi and Insects
Authors: D'Ovidio Renato , Raiola Alessandro , Capodicasa Cristina , Devoto Alessandra , Pontiggia Daniela , Roberti Serena , Galletti Roberta , Conti Eric , O'Sullivan Donal , De Lorenzo Giulia
Published: August 2004

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.
Tobacco Mosaic Virus Regulates the Expression of Its Own Resistance Gene N
Authors: Levy Michal , Edelbaum Orit , Sela Ilan
Published: August 2004

The N gene of tobacco (Nicotiana tabacum) is a typical resistance (R) gene engendering localization of tobacco mosaic virus (TMV) infection and the elicitation of a hypersensitive necrotic response. The consensus model for R gene-derived resistance is at the level of protein:protein interactions, in which proteins of the pathogen interact with already present receptor-like proteins produced by the plant's R genes. This article demonstrates, by quantitative real-time reverse transcription-PCR analysis, that in tobacco carrying the dominant allele N, a basal level of transcription indeed occurs in noninfected plants. However, accumulation of N-mRNA in infected plants indicates that transcription is stimulated by TMV infection (up to 38-fold in locally infected leaves and up to 165-fold in upper, noninoculated leaves). Potato virus Y infection did not result in accumulation of N-mRNA, indicating a specific TMV-related phenomenon. The possible uncoupling of viral restriction from necrosis is discussed.
Novel Regulation of Aquaporins during Osmotic Stress
Authors: Vera-Estrella Rosario , Barkla Bronwyn J. , Bohnert Hans J. , Pantoja Omar
Published: August 2004

Aquaporin protein regulation and redistribution in response to osmotic stress was investigated. Ice plant (Mesembryanthemum crystallinum) McTIP1;2 (McMIPF) mediated water flux when expressed in Xenopus leavis oocytes. Mannitol-induced water imbalance resulted in increased protein amounts in tonoplast fractions and a shift in protein distribution to other membrane fractions, suggesting aquaporin relocalization. Indirect immunofluorescence labeling also supports a change in membrane distribution for McTIP1;2 and the appearance of a unique compartment where McTIP1;2 is expressed. Mannitol-induced redistribution of McTIP1;2 was arrested by pretreatment with brefeldin A, wortmannin, and cytochalasin D, inhibitors of vesicle trafficking-related processes. Evidence suggests a role for glycosylation and involvement of a cAMP-dependent signaling pathway in McTIP1;2 redistribution. McTIP1;2 redistribution to endosomal compartments may be part of a homeostatic process to restore and maintain cellular osmolarity under osmotic-stress conditions.
Understanding in Vivo Benzenoid Metabolism in Petunia Petal Tissue
Authors: Boatright Jennifer , Negre Florence , Chen Xinlu , Kish Christine M. , Wood Barbara , Peel Greg , Orlova Irina , Gang David , Rhodes David , Dudareva Natalia
Published: August 2004

In vivo stable isotope labeling and computer-assisted metabolic flux analysis were used to investigate the metabolic pathways in petunia (Petunia hybrida) cv Mitchell leading from Phe to benzenoid compounds, a process that requires the shortening of the side chain by a C2 unit. Deuterium-labeled Phe (2H5-Phe) was supplied to excised petunia petals. The intracellular pools of benzenoid/phenylpropanoid-related compounds (intermediates and end products) as well as volatile end products within the floral bouquet were analyzed for pool sizes and labeling kinetics by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Modeling of the benzenoid network revealed that both the CoA-dependent, β-oxidative and CoA-independent, non-β-oxidative pathways contribute to the formation of benzenoid compounds in petunia flowers. The flux through the CoA-independent, non-β-oxidative pathway with benzaldehyde as a key intermediate was estimated to be about 2 times higher than the flux through the CoA-dependent, β-oxidative pathway. Modeling of 2H5-Phe labeling data predicted that in addition to benzaldehyde, benzylbenzoate is an intermediate between l-Phe and benzoic acid. Benzylbenzoate is the result of benzoylation of benzyl alcohol, for which activity was detected in petunia petals. A cDNA encoding a benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase was isolated from petunia cv Mitchell using a functional genomic approach. Biochemical characterization of a purified recombinant benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase protein showed that it can produce benzylbenzoate and phenylethyl benzoate, both present in petunia corollas, with similar catalytic efficiencies.
Characterization of the Arabidopsis Lysine-Rich Arabinogalactan-Protein AtAGP17 Mutant (rat1) That Results in a Decreased Efficiency of Agrobacterium Transformation
Authors: Gaspar Yolanda Maria , Nam Jaesung , Schultz Carolyn Jane , Lee Lan-Ying , Gilson Paul R. , Gelvin Stanton B. , Bacic Antony
Published: August 2004

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17. We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17. Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants. Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.