bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolite profiling of plant carotenoids using the matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry
Authors: Fraser Paul D. , Enfissi Eugenia M. A. , Goodfellow Michael , Eguchi Tadashi , Bramley Peter M.
Published: January 2007

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces ‘spot to spot’ variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi‐quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC‐photodiode array analysis. These data show that MALDI/TOF‐MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.
Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana
Authors: Qian Weiqiang , Yu Chunmei , Qin Huanju , Liu Xin , Zhang Aimin , Johansen Ida Elisabeth , Wang Daowen
Published: January 2007

Phosphomannomutase (PMM) catalyzes the interconversion of mannose‐6‐phosphate and mannose‐1‐phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53‐6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine‐tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose‐1‐phosphate into mannose‐6‐phosphate and glucose‐1‐phosphate into glucose‐6‐phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus‐induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral‐vector‐mediated ectopic expression led to a 20–50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM–GFP fusion protein in Arabidopsis also increased AsA content by 25–33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.
The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation
Authors: Roldán Isaac , Wattebled Fabrice , Mercedes Lucas M. , Delvallé David , Planchot Veronique , Jiménez Sebastian , Pérez Ricardo , Ball Steven , D'Hulst Christophe , Mérida Ángel
Published: January 2007

All plants and green algae synthesize starch through the action of the same five classes of elongation enzymes: the starch synthases. Arabidopsis mutants defective for the synthesis of the soluble starch synthase IV (SSIV) type of elongation enzyme have now been characterized. The mutant plants displayed a severe growth defect but nonetheless accumulated near to normal levels of polysaccharide storage. Detailed structural analysis has failed to yield any change in starch granule structure. However, the number of granules per plastid has dramatically decreased leading to a large increase in their size. These results, which distinguish the SSIV mutants from all other mutants reported to date, suggest a specific function of this enzyme class in the control of granule numbers. We speculate therefore that SSIV could be selectively involved in the priming of starch granule formation.
Single‐stranded DNA binding factor AtWHY1 modulates telomere length homeostasis in Arabidopsis
Authors: Yoo Hyun Hee , Kwon Chian , Lee Myeong Min , Chung In Kwon
Published: January 2007

Telomere homeostasis, a process that is essential for the maintenance of chromosome integrity, is regulated by telomerase and a collection of associated proteins. By mass spectrometry we have identified a new telomeric protein encoded by the AtWHY1 (Arabidopsis thaliana Whirly 1) gene in Arabidopsis. AtWHY1 specifically binds the single‐stranded plant telomeric DNA sequences, but not double‐stranded telomeric DNA. To gain insights into the function of AtWHY1 in telomere biogenesis, we have identified two Arabidopsis lines harboring T‐DNA insertions in AtWHY1. These lines exhibit neither growth nor developmental defects. However, AtWHY1‐deficient plants show a steady increase in the length of telomere tracts over generations. This telomere elongation is correlated with a significant increase in telomerase activity. On the contrary, transgenic plants expressing AtWHY1 show a decreased telomerase activity and shortened telomeres. The evidence presented here indicates that AtWHY1 is a new family of telomere end‐binding proteins that plays a role in regulating telomere‐length homeostasis in Arabidopsis.
Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers
Authors: Carlsson Jenny , Lagercrantz Ulf , Sundström Jens , Teixeira Rita , Wellmer Frank , Meyerowitz Elliot M. , Glimelius Kristina
Published: January 2007

To gain new insights into the mechanism underlying cytoplasmic male sterility (CMS), we compared the nuclear gene expression profiles of flowers of a Brassica napus CMS line with that of the fertile B. napus maintainer line using Arabidopsis thaliana flower‐specific cDNA microarrays. The CMS line used has a B. napus nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of both B. napus and A. thaliana DNA. Gene expression profiling revealed that a large number of genes differed in expression between the two lines. For example, nuclear genes coding for proteins that are involved in protein import into organelles, genes expressed in stamens and pollen, as well as genes implicated in either cell‐wall remodeling or architecture, were repressed in the CMS line compared with B. napus. These results show that the mt genome of the CMS line strongly influences nuclear gene expression, and thus reveal the importance of retrograde signalling between the mitochondria and the nucleus. Furthermore, flowers of the CMS line are characterized by a replacement of stamens with carpelloid organs, and thus partially resemble the APETALA3 (AP3) and PISTILLATA (PI) mutants. In accordance with this phenotype, AP3 expression was downregulated in the stamens, shortly before these organs developed carpelloid characteristics, even though it was initiated correctly. Repression of PI succeeded that of AP3 and might be a consequence of a loss of AP3 activity. These results suggest that AP3 expression in stamens depends on proper mt function and a correct nuclear–mt interaction, and that mt alterations cause the male sterility phenotype of the CMS line.
The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein
Authors: Lundin Björn , Hansson Maria , Schoefs Benoît , Vener Alexander V. , Spetea Cornelia
Published: January 2007

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water‐oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T‐DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2‐deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady‐state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild‐type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild‐type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.
Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon‐deprived Arabidopsis seedlings
Authors: Osuna Daniel , Usadel Bjorn , Morcuende Rosa , Gibon Yves , Bläsing Oliver E. , Höhne Melanie , Günter Manuela , Kamlage Beate , Trethewey Richard , Scheible Wolf‐Rüdiger , Stitt Mark
Published: January 2007

Arabidopsis seedlings were subjected to 2 days of carbon starvation, and then resupplied with 15 mm sucrose. The transcriptional and metabolic response was analyzed using ATH1 arrays, real‐time quantitative (q)RT‐PCR analysis of >2000 transcription regulators, robotized assays of enzymes from central metabolism and metabolite profiling. Sucrose led within 30 min to greater than threefold changes of the transcript levels for >100 genes, including 20 transcription regulators, 15 ubiquitin‐targeting proteins, four trehalose phosphate synthases, autophagy protein 8e, several glutaredoxins and many genes of unknown function. Most of these genes respond to changes of endogenous sugars in Arabidopsis rosettes, making them excellent candidates for upstream components of sugar signaling pathways. Some respond during diurnal cycles, consistent with them acting in signaling pathways that balance the supply and utilization of carbon in normal growth conditions. By 3 h, transcript levels change for >1700 genes. This includes a coordinated induction of genes involved in carbohydrate synthesis, glycolysis, respiration, amino acid and nucleotide synthesis, DNA, RNA and protein synthesis and protein folding, and repression of genes involved in amino acid and lipid catabolism, photosynthesis and chloroplast protein synthesis and folding. The changes of transcripts are followed by a delayed activation of central metabolic pathways and growth processes, which use intermediates from these pathways. Sucrose and reducing sugars accumulate during the first 3–8 h, and starch for 24 h, showing that there is a delay until carbon utilization for growth recommences. Gradual changes of enzyme activities and metabolites are found for many metabolic pathways, including glycolysis, nitrate assimilation, the shikimate pathway and myoinositol, proline and fatty acid metabolism. After 3–8 h, there is a decrease of amino acids, followed by a gradual increase of protein.
Dissecting the biosynthetic pathway for the bypass1 root‐derived signal
Authors: Van Norman Jaimie M. , Sieburth Leslie E.
Published: January 2007

The Arabidopsis BYPASS1 (BPS1) gene is required for normal root and shoot development. In bps1 mutants, grafting and root excision experiments have shown that mutant roots produce a transmissible signal that is capable of arresting shoot development. In addition, we previously showed that growth of bps1 mutants on the carotenoid biosynthesis inhibitor fluridone resulted in partial rescue of both leaf and root defects. These observations suggest that a single mobile carotenoid‐derived signal affects both root and shoot development. Here, we describe further characterization of the bps1 root‐derived signal using genetic and biosynthetic inhibitor approaches. We characterized leaf and root development in double mutants that combined the bps1 mutant with mutants that have known defects in genes encoding carotenoid processing enzymes or defects in responses to carotenoid‐derived abscisic acid. Our studies indicate that the mobile signal is neither abscisic acid nor the MAX‐dependent hormone that regulates shoot branching, and that production of the signal does not require the activity of any single carotenoid cleavage dioxygenase. In addition, our studies with CPTA, a lycopene cyclase inhibitor, show that signal production requires synthesis of β‐carotene and its derivatives. Furthermore, we show a direct requirement for carotenoids as signal precursors, as the GUN plastid‐to‐nucleus signaling pathway is not required for phenotypic rescue. Together, our results suggest that bps1 roots produce a novel mobile carotenoid‐derived signaling compound.
The hyper‐fluorescent trichome phenotype of the brt1 mutant of Arabidopsis is the result of a defect in a sinapic acid:UDPG glucosyltransferase
Authors: Sinlapadech Taksina , Stout Jake , Ruegger Max O. , Deak Michael , Chapple Clint
Published: January 2007

Sinapoylmalate is a major phenylpropanoid that is accumulated in Arabidopsis. Its presence causes the adaxial surface of leaves to fluoresce blue under UV light, and mutations that lead to lower levels of sinapoylmalate decrease UV‐induced leaf fluorescence. The Arabidopsis bright trichomes 1 (brt1) mutant was first identified in a screen for mutants that exhibit a reduced epidermal fluorescence phenotype; however, subsequent examination of the mutant revealed that its trichomes are hyper‐fluorescent. The results from genetic mapping and complementation analyses showed that BRT1 (At3g21560) encodes UGT84A2, a glucosyltransferase previously shown to be capable of using sinapic acid as a substrate. Residual levels of sinapoylmalate and sinapic acid:UDP‐glucose glucosyltransferase activity in brt1 leaves suggest that BRT1 is one member of a family of partially redundant glycosyltransferases that function in Arabidopsis sinapate ester biosynthesis. RT‐PCR analysis showed that BRT1 is expressed through all stages of plant life cycle, a result consistent with the impact of the brt1 mutation on both leaf sinapoylmalate levels and seed sinapoylcholine content. Finally, the compound accumulated in brt1 trichomes was identified as a sinapic acid‐derived polyketide, indicating that when sinapic acid glycosylation is reduced, a portion of it is instead activated to its CoA thioester, which then serves as a substrate for chalcone synthase.
A lysine‐rich arabinogalactan protein in Arabidopsis is essential for plant growth and development, including cell division and expansion
Authors: Yang Jie , Sardar Harjinder S. , McGovern Kathleen R. , Zhang Yizhu , Showalter Allan M.
Published: January 2007

Arabinogalactan proteins (AGPs), a family of hydroxyproline‐rich glycoproteins, occur throughout the plant kingdom. The lysine‐rich classical AGP subfamily in Arabidopsis consists of three members, AtAGP17, 18 and 19. In this study, AtAGP19 was examined in terms of its gene expression pattern and function. AtAGP19 mRNA was abundant in stems, with moderate levels in flowers and roots and low levels in leaves. AtAGP19 promoter‐controlled GUS activity was high in the vasculature of leaves, roots, stems and flowers, as well as styles and siliques. A null T‐DNA knockout mutant of AtAGP19 was obtained and compared to wild‐type (WT) plants. The atagp19 mutant had: (i) smaller, rounder and flatter rosette leaves, (ii) lighter‐green leaves containing less chlorophyll, (iii) delayed growth, (iv) shorter hypocotyls and inflorescence stems, and (v) fewer siliques and less seed production. Several abnormalities in cell size, number, shape and packing were also observed in the mutant. Complementation of this pleiotropic mutant with the WT AtAGP19 gene restored the WT phenotypes and confirmed that AtAGP19 functions in various aspects of plant growth and development, including cell division and expansion, leaf development and reproduction.
1.5 Mb de novo 22q11.21 microduplication in a patient with cognitive deficits and dysmorphic facial features
Authors: Alberti A , Romano C , Falco M , Calì F , Schinocca P , Galesi O , Spalletta A , Di Benedetto D , Fichera M
Published: January 2007

The 22q11.2 microduplication syndrome is caused by non‐allelic homologous recombination mediated by misalignments of low copy repeats located in the region deleted in the DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). The variable phenotype of such condition, consisting in a combination of dysmorphic facial features, cognitive deficits, velopharyngeal insufficiency, congenital heart defects and immunologic derangement, is caused usually in 90% of cases by a 3 Mb deletion or in a minority of cases (7%) by a 1.5 Mb deletion. The most common reciprocal event of deletion is the 3 Mb duplication, reported more recently with a variable phenotype, ranging from multiple defects to normality. In this study, we report a 2.5‐year‐old girl with cognitive deficits and dysmorphic facial features such as superior placement of eyebrows, upslanting palpebral fissures, widely spaced eyes, broad nasal bridge and epicanthal folds. Fluorescent in situ hybridization for DGS/VCFS region on metaphase chromosomes did not show any apparent anomaly. Subsequent array comparative genomic hybridization study, confirmed by multiplex ligation‐dependent probe assay and microsatellite analysis, disclosed a 1.5 Mb de novo 22q11.21 duplication concerning the same chromosomal region deleted in a minority of patients with DGS. These findings identify the minimal duplicated region leading to this emerging syndrome.
A new catalytic activity from tobacco converting 2‐coumaric acid to salicylic aldehyde
Authors: Malinowski Jacek , Krzymowska Magdalena , Godoń Kamil , Hennig Jacek , Podstolski Andrzej
Published: January 2007

Salicylic acid (SA) mediates plant response to pathogen invasion, resulting in hypersensitive response and in the formation of systemic acquired resistance. It is well known that Nicotiana tabacum and other plants respond to Tobacco Mosaic Virus (TMV) infection by increasing the content of SA but the details of SA biosynthesis are still not fully understood. Generally, SA may originate directly from isochorismate (Arabidopsis thaliana), or its C6–C1 skeleton could be synthesized via the phenylpropanoid pathway by β‐oxidation of trans‐cinnamic acid (N. tabacum), 2‐coumaric acid (OCA) (Gaulteria procumbens, Lycopersicum esculentum) or by retro‐aldol reaction of trans‐cinnamoyl‐CoA (Hypericum androsaemum). We report here a novel putative enzyme activity from tobacco, salicylic aldehyde synthase (SAS), catalysing non‐oxidative formation of salicylic aldehyde (SALD) directly from OCA. This chain‐shortening activity is similar to that of 4‐hydroxybenzaldehyde synthase from Vanilla planifolia, Lithospermum erythrorhizon, Daucus carota, Solanum tuberosum and Polyporus hispidus but the enzyme differs in the kinetics of the reaction, substrate specificity and requirements for reducing cofactors. SAS activity is constitutively expressed in healthy tobacco leaves and doubles as a result of infection with TMV. Moreover, the product of SAS activity—SALD, applied exogenously on tobacco leaves, stimulates peroxidase activity and enhances resistance to consecutive infection with TMV. These observations could suggest a contribution of SAS and SALD to the response of tobacco to TMV infection.
Induction of enhanced tolerance to cold stress and disease by overexpression of the pepper CaPIF1 gene in tomato
Authors: Seong Eun Soo , Baek Kwang‐Hyun , Oh Sang‐Keun , Jo Sung Hwan , Yi So Young , Park Jeong Mee , Joung Young Hee , Lee Sanghyeob , Cho Hye Sun , Choi Doil
Published: January 2007

We previously isolated CaPIF1 (Capsicum annuum Pathogenesis Induced Factor) from pepper leaves following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. The CaPIF1 gene encodes a plant‐specific Cys‐2/His‐2 zinc finger protein that functions as a transcription factor. To elucidate the role of the CaPIF1 transcription factor in gene regulation, we transformed tomato plants expressing full‐length CaPIF1 and conducted microarray experiments. The ectopic overexpression of pepper CaPIF1 in tomato resulted in a massive change in gene expression: of a total of 8700 genes on the microarray, 110 were up‐ or downregulated, without exhibiting any visual morphological abnormality. The microarray analysis showed that CaPIF1 turned on genes involved in metabolic pathways, and stress and defense responses; the results also suggested that the upregulation of the genes might increase tolerance to cold stress and pathogen attack. The transgenic tomato displayed more tolerance to cold stress and to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000, and the tolerance levels were correlated with CaPIF1 expression levels. These results suggest that CaPIF1 is a unique Cys‐2/His‐2 transcription factor, regulating multiple genes both directly and indirectly, and thus conferring increased tolerance to biotic and abiotic stresses.
The properties of the Mn, Ni and Pb transport operating at plasma membranes of cucumber roots
Authors: Migocka Magdalena , Klobus Grazyna
Published: January 2007

To avoid metal toxicity, plants have developed mechanisms including efflux of metal ions from cells and their sequestration into cellular compartments. In this report, we present evidence for the role of plasma membrane efflux systems in metal tolerance of cucumber roots. We have identified the plasma membrane‐transport system participating in Cd, Pb, Mn and Ni efflux from the cytosol. Kinetic characterization of this proton‐coupled transport system revealed that it is saturable and has a different affinity for each of the metal ions used (with Km 5, 7.5 and 0.1 mM for Mn, Ni and Pb, respectively). Treatment of cucumber roots with 100 μM Cd prior to the transport measurements caused a great increase (over 250%) in Cd antiport activity in plasmalemma vesicles. After decreasing the metal concentration to 50 μM we still observed a large increase (over 150%) of this activity in comparison with the control. Moreover, the addition of 50 μM Cd to the external solution stimulated not only Cd antiport in the plasmalemma vesicles but also the antiport of other metals used in the experiments. Treatment of cucumber roots with 50 μM Ni revealed a similar effect: the antiport activity of Cd, Mn, Ni and Pb was stimulated, although to a lesser extent in comparison with stimulation by Cd. The data indicate that the root plasma membrane antiporter system is stimulated by the exogenous presence of heavy metals.
Catalase inhibition alters suberization and wound healing in potato (Solanum tuberosum) tubers
Authors: Bajji Mohammed , M’Hamdi Mahmoud , Gastiny Frédéric , Delaplace Pierre , Fauconnier Marie‐Laure , Du Jardin Patrick
Published: January 2007

In response to wounding, potato (Solanum tuberosum L.) tubers generate hydrogen peroxide (H2O2) in association with suberization, a critical phase of the wound‐healing process. In the present study, the effect of aminotriazole (AT), a catalase (CAT, EC 1.11.1.6) inhibitor, on cut tubers was investigated using fresh weight (FW) loss and pathogen attack symptoms as indicators of wound‐healing efficiency. Seven days after treatment, AT‐treated tuber halves lost more FW and developed infection signs compared with the controls. Thiourea, another CAT inhibitor, as well as exogenous H2O2 treatments induced the same effects as AT suggesting that the alteration of the wound healing may be caused by CAT inhibition and the resulting accumulation of H2O2. Using transgenic tubers, FW losses 1 week after wounding were either higher (CAT repression) or lower (CAT overexpression) than those of the wild‐type. When tuber halves were allowed to wound heal for different periods before treatment, AT had no effect on the progress of their wound healing if wound‐healed for at least 3 days. This implies that AT may affect early wound‐healing‐related events, especially those occurring before or during suberization. A time‐course analysis of the effects of AT treatment on wounded tuber tissues revealed that AT prevented the deposition of the polyphenolic domain of suberin in association with CAT inhibition and H2O2 accumulation. These data are important in identifying factors that may be required to regulate suberization and contribute to a better understanding of this critical process to hasten its rate and limit wound‐related losses in stored potato tubers.
Rapid breakdown of exogenous extracellular hydrogen peroxide by lichens
Authors: Beckett Richard P. , Minibayeva Farida V.
Published: January 2007

All organisms, even highly stress‐tolerant lichens, produce a variety of reactive oxygen species (ROS) during and after stress. Furthermore, the cell walls of some lichens in Suborder Peltigerineae contain laccases, and therefore can produce quinone radicals that can break down to yield ROS. While the extracellular ROS produced by these enzymes probably play important roles in the biology of these lichens, they may also be potentially harmful and need to be rapidly broken down. To test this, rates of breakdown of exogenously supplied H2O2 were measured in a range of lichen species. Considerable diversity existed in rates of H2O2 breakdown but rates were on average almost double in members of Suborder Peltigerineae. While all lichens tested appeared to lack extracellular peroxidases and catalases, enzymes normally involved in breaking down H2O2, extracellular tyrosinase activity could be readily detected in the Peltigerineae. A role for tyrosinases in H2O2 breakdown was supported by the results from experiments involving inhibitors, and demonstration of the simultaneous release into an incubation solution of tyrosinase activity and the ability to breakdown H2O2. Rates of breakdown were very high, and tyrosinase appeared to break down H2O2 by a catalase‐like mechanism. However, significant rates of breakdown of H2O2 also occurred in species that did not possess cell wall redox enzymes. These species probably took up the exogenously supplied H2O2 intracellularly and then broke it down by the usual catalases and peroxidases. The importance of H2O2 degradation is discussed in terms of its possible role in defence against the harmful effects of ROS.
Seasonal changes in the ion‐mediated increase of xylem hydraulic conductivity in stems of three evergreens: any functional role?
Authors: Gascó Antonio , Salleo Sebastiano , Gortan Emmanuelle , Nardini Andrea
Published: January 2007

Changes in hydraulic conductivity (Kh) were measured monthly in stems of Laurus nobilis between July 2005 and June 2006 and in March and June 2006 in stems of Prunus laurocerasus and Phillyrea latifolia. Percent enhancement of Kh (ΔKh) was recorded as KCl injected into stems compared with deionized water. Stems of L. nobilis perfused with increasing [KCl] saturated the hydraulic effect at 50 mM KCl, while P. laurocerasus and Ph. latifolia showed saturation at 17 and less than 5 mM KCl, respectively. Impressive seasonal changes in ΔKh were recorded in L. nobilis, from +120% in February to +20% during the growth period. ΔKh was negatively correlated to minimum air temperatures and was highest during winter frost. Negative correlation also existed between ΔKh and conduit lengths as detected during transition of plants from activity to winter rest and from winter to spring. In the winter, the thermal effect was prevailing. High cavitation‐induced Kh loss coincided with high ΔKh. Similar but much smaller seasonal effects on ΔKh were recorded in the other two species where, however, also smaller seasonal changes in conduit lengths were recorded. We conclude that (1) the K+‐induced effect on Kh was because of interference of the cation with pectins of intervessel pit membranes (increasingly crossed by the ionic solution in stems with shorter conduits); (2) winter frost might have an effect on ΔKh and (3) seasonal changes in ΔKh because of possible change in pectic composition may represent a mechanism to regulate water flows in planta.
Isolation and characterization of carbonic anhydrases from the marine diatom Phaeodactylum tricornutum
Authors: Szabo Eva , Colman Brian
Published: January 2007

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CAext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO2‐grown cells. CAext was detected only in UTEX 640 grown under CO2‐limited conditions and present in trace amounts in cells grown on high CO2. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster‐running band which was absent from extracts of high CO2‐grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CAext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low‐CO2‐inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO2‐grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air‐grown cells indicated that the CAext was located in the periplasmic space and on the cell membrane, whereas in high CO2‐grown cells only internal CA was detected.
Some photosynthetic responses to salinity resistance are transferred into the somatic hybrid descendants from the wild soybean Glycine cyrtoloba ACC547
Authors: Yang Yong , Yan Cheng‐Qi , Cao Bao‐Hua , Xu Hong‐Xia , Chen Jian‐Ping , Jiang De‐An
Published: January 2007

The somatic hybrid descendants between a cultivated soybean Glycine max Melrose and a wild species Glycine cyrtoloba ACC547 were found to possess some salinity‐resistant traits of the wild soybean. Under salt stress, two of the descendants as well as their wild parent grew better than their cultivated parent. In addition, salinity‐induced decline in the net photosynthetic rate and the maximum photochemical efficiency was much less in the wild species and the descendants than in Melrose when stressed for more than 5 days. Analysis of the postillumination transient increase in chlorophyll fluorescence and the dark rereduction of the oxidized primary electron donor in photosystem I (PSI) (P700+) indicated that salinity induced a significant upregulation of the cyclic electron flow around PSI (CEF1) in the wild species and the hybrid descendants. Similar to their wild parent, the descendants maintained higher non‐photochemical dissipation of excess excitation energy than their cultivated parent under salt stress. As a consequence, there were lower levels of superoxide radical and membrane lipid peroxidation in the plants of the descendants and the wild species. Based on these results, we proposed that the high salinity resistance of the descendants might be because of, at least partially, the trait inherited from the wild species of the enhanced CEF1 which contributed to the sufficient dissipation of excess excitation energy to protect photosynthetic apparatus from the damage of reactive oxygen species.
Limiting cytosolic Na+ confers salt tolerance to rice cells in culture: a two‐photon microscopy study of SBFI‐loaded cells
Authors: Anil Veena S. , Krishnamurthy H. , Mathew M. K.
Published: January 2007

Rice (Oryza sativa L.), a staple food in Asia, is very sensitive to soil salinity. However, intraspecific variations exist, with the coastal cultivar Pokkali tolerating even brackish water. This study explores cellular mechanisms that contribute to salt tolerance in rice. It is widely accepted that limiting cytosolic Na+ should improve the survival of plants subjected to saline stress. However, an understanding of the mechanisms by which Na+ levels are controlled in relatively tolerant cultivars requires monitoring cytosolic Na+ non‐invasively and in real time, which is technically challenging. We have used two‐photon excitation for the ratiometric estimation of cytosolic Na+ in cultured cells using sodium‐binding benzofuran isophthalate. Pokkali cells maintained low cytosolic Na+ (approximately 25 mM), and a viability of over 85% under high salinity , while Jaya cells were unable to maintain low cytosolic Na+ and suffered decreased viability even at moderate saline stress. Here we show that the permeability of the Pokkali plasma membrane to Na+ is significantly lower than that of Jaya, to the extent that it is comparable with permeabilities reported for halophytes. Pokkali effectively sequesters Na+ in intracellular compartments utilizing a Ca2+‐regulated transport system(s). Together these cellular mechanisms allow Pokkali to maintain low cytosolic Na+ up to a stress of 250 mM NaCl. The findings demonstrate that differences in survival between these contrasting varieties of rice are mainly because of differences in membrane transport mechanisms and thus have significance in crop improvement.
Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster‐to‐rat heart xenotransplantation
Authors: Séveno Céline , Coulon Flora , Haspot Fabienne , Mérieau Emmanuel , Renaudin Karine , Martinet Bernard , Vanhove Bernard
Published: January 2007

Abstract:  Background:  In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T‐cell activation and promotes a long‐term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4+T cell‐mediated xenograft rejection.

Methods:  Hamster hearts were transplanted into LEW.1A rats receiving an accommodation‐inducing treatment consisting of a short course administration of LF15‐0195 and a daily administration of cyclosporine A (CSA). To achieve long‐term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad‐CTLA4Ig) was added to the accommodation induction protocol. On day 40 post‐transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti‐CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined.

Results:  Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4+ xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad‐CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non‐donor‐specific suppressive activity dependent on IL‐2, NO, and IDO. These regulatory cells were different from those previously identified after Ad‐CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti‐donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival.

Conclusion:  CD28/B7 blockade delays CD4+ T cell‐mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement‐dependent manner.
Coagulation cascade activation triggers early failure of pig hearts expressing human complement regulatory genes
Authors: Wu Guosheng , Pfeiffer Steffen , Schröder Carsten , Zhang Tianshu , Nguyen Bao N. , Kelishadi Sean , Atkinson James B. , Schuurman Henk‐Jan , White David J.G. , Azimzadeh Agnes M. , Pierson Richard N.
Published: January 2007

Abstract:  Background:  Hyperacute rejection (HAR) and early graft failure (EGF) have been described in a minority of pig‐to‐baboon heart transplants using organs transgenic for human complement regulatory proteins (hCRP). Here we investigate the role of coagulation cascade activation in the pathogenesis of HAR and EGF in a consecutive series where a high incidence of these outcomes was observed.

Methods:  Twenty‐eight naïve wild‐caught Papio anubis baboons received heterotopic heart transplants from pigs transgenic for hDAF (n = 23) or hMCP (n = 5). Immunosuppression consisted of cyclosporine A, cyclophosphamide and MMF (n = 18) or anti‐CD154 mAb (IDEC‐131) and ATG (n = 10). Eleven received anti‐Gal carbohydrates (GAS914, n = 8, or NEX1285, n = 3), of which four also underwent extracorporeal immunoadsorption (EIA), and 12 also received pharmacologic complement inhibitors (C1 INH, n = 9, or APT070, n = 3).

Results:  Excluding one technical failure, 14 of 27 transplants (11 hDAF, 3 hMCP) exhibited either HAR (n = 10) or EGF (n = 4). Surprisingly, neither complement inhibition (with C1 INH or APT070) nor anti‐Gal antibody depletion with GAS914, NEX1285, or additional EIA consistently prevented HAR or EGF despite low or undetectable complement deposition. Strikingly, most grafts with HAR/EGF exhibited prominent fibrinogen and platelet deposition associated with systemic coagulation cascade activation, consistent with non‐physiologic intravascular coagulation, in many instances despite little evidence for antibody‐mediated complement activation.

Conclusion:  We conclude that dysregulated coagulation correlates closely with and probably causes primary failure of pig hearts transgenic for hCRP. These data support efforts to define effective strategies to prevent dysregulated coagulation in pig organ xenografts.
Chemokine and toll‐like receptor signaling in macrophage mediated islet xenograft rejection
Authors: Chandra Abhilash P. , Ouyang Li , Yi Shounan , Wong Jeffrey K. W. , Ha Hong , Walters Stacey N. , Patel Anita T. , Stokes Rebecca , Jardine Meg , Hawthorne Wayne J. , O'Connell Philip J.
Published: January 2007

Abstract:  Background:  Adoptive transfer of antigen‐primed T‐cell‐activated macrophages into NOD‐SCID mice within 14 days of foetal porcine pancreatic fragment (FPP) or foetal porcine skin (FPS) transplantation had been shown to cause xenograft rejection. In the present study, it was proposed that signaling between the graft and macrophages promoted specific graft recognition and destruction in this setting.

Methods:  Exogenous macrophages isolated from rejecting FPP xenografts were transferred to NOD‐SCID FPP recipients and tracked by Ly5.1 surface antigen or via CSFE staining. Monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), macrophage inflammatory protein‐1β (MIP‐1β), regulated upon activation, normal T‐cell expressed and secreted (RANTES), chemokine (C‐C motif) receptor 2 (CCR2), chemokine (C‐C motif) receptor 5 (CCR5), toll‐like receptors (TLRs) (1–9) and gene expression in transplanted FPP xenografts was evaluated by real‐time polymerase chain reaction. Gene expression of CCR2, CCR5 and TLRs was also analyzed in pooled samples of activated and non‐activated macrophages.

Results:  Exogenous macrophages were shown to track to and reject recently transplanted but not established FPP xenografts. Gene expression for MCP‐1, RANTES, MIP‐1α and MIP‐1β was at least 3‐fold greater in recently transplanted compared with established xenografts (P < 0.05), and CCR2 and CCR5 gene expression was 10‐fold greater in activated compared non‐activated macrophages, suggesting that graft‐mediated pro‐inflammatory signals were important for macrophage recruitment. Specific graft recognition by macrophages may involve TLR signaling as macrophages exposed to porcine islets had higher levels of TLR gene expression compared with those exposed to allografts regardless of the level of activation.

Conclusion:  Xenografts provide additional activation signals to macrophages that are not seen following allotransplantation. This study identifies chemokines and TLR as important signals in macrophage‐mediated recognition and rejection of islet xenografts.
Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs
Authors: Kim Jae Hyeon , Kim Hyoung‐Il , Lee Kang‐Woo , Yu Jae Eun , Kim Sun Hee , Park Ho Sun , Park Chung‐Gyu , Ihm Sung‐Hee , Ha Jongwon , Kim Sang Joon , Lee Hong Kyu , Ahn Curie , Park Kyong Soo
Published: January 2007

Abstract:  Background:  Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen‐free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse.

Methods:  Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6–7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation.

Results:  The islet yield per gram of pancreas from ACM pigs (9589 ± 2823 IEQ/g) was significantly higher than that from APM pigs (1752 ± 874 IEQ/g, P < 0.05), AM pigs (1931 ± 947 IEQ/g, P < 0.05), or YCM pigs (3460 ± 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs.

Conclusions:  We isolated extremely high yields of well‐functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.
Coagulation and the xenograft endothelium
Authors: Cowan Peter J.
Published: January 2007

Abstract:  Acute humoral rejection remains the major barrier to long‐term pig‐to‐primate xenograft survival, and microvascular thrombosis is a critical element of the rejection process. It appears that persistent endothelial cell activation and injury, by even low levels of anti‐graft antibodies, eventually overwhelm the cellular anticoagulant defences and promote the development of thrombotic microangiopathy. Porcine endothelium may be particularly vulnerable because of cross‐species molecular incompatibilities affecting the function of thrombomodulin and possibly TFPI. Recent data from small animal models suggest that transgenic overexpression of anti‐thrombotic molecules on xenograft endothelium is capable of inhibiting intravascular thrombosis and preventing acute humoral rejection. In conjunction with existing genetic modifications (e.g. Gal KO, hDAF), this is a promising strategy to move xenotransplantation to the clinic.
Short‐ and long‐term benefits and detriments to recombination under antagonistic coevolution
Authors: PETERS A. D. , LIVELY C. M.
Published: January 2007

We explored the evolution of recombination under antagonistic coevolution, concentrating on the equilibrium frequencies of modifier alleles causing recombination in initially nonrecombining populations. We found that the equilibrium level of recombination in the host depended not only on parasite virulence, but also on the strength of the modifier allele, and on whether or not the modifier was physically linked to the parasite interaction loci. Nonetheless, the maximum level of recombination for linked loci at equilibrium was about 0.3 (60% of free recombination) for interactions with highly virulent parasites; the level decreased for unlinked modifiers, and for lower levels of parasite virulence. We conclude that recombination spreads because it provides a combination of an immediate (next‐generation) fitness benefit and a delayed (two or more generations) increase in the rate of response to directional selection. The relative impact of these two mechanisms depends on the virulence of parasites early in the spread of the modifier, but a trade‐off between the two dictates the equilibrium modifier frequency for all nonzero virulences that we examined. In addition, population mean fitness was higher in populations at intermediate equilibria than populations fixed for free recombination or no recombination. The difference, however, was not enough on its own to overcome the two‐fold cost of producing males.
Cope's Rule in the Pterosauria, and differing perceptions of Cope's Rule at different taxonomic levels
Authors: HONE D. W. E. , BENTON M. J.
Published: January 2007

The remarkable extinct flying reptiles, the pterosaurs, show increasing body size over 100 million years of the Late Jurassic and Cretaceous, and this seems to be a rare example of a driven trend to large size (Cope's Rule). The size increases continue throughout the long time span, and small forms disappear as larger pterosaurs evolve. Mean wingspan increases through time. Examining for Cope's Rule at a variety of taxonomic levels reveals varying trends within the Pterosauria as a whole, as pterodactyloid pterosaurs increase in size at all levels of examination, but rhamphorhynchoid pterosaurs show both size increase and size decrease in different analyses. These results suggest that analyses testing for Cope's Rule at a single taxonomic level may give misleading results.