Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Assessing the use of frozen pork meat in the manufacture of cooked ham

This study aimed to evaluate the effects of slow (–18 °C) and fast freezing (liquid nitrogen) of pork meat, and the use of exudate released upon thawing, on the physicochemical, color, rheological, microbiological, histological, and sensory characteristics of cooked ham. The meat samples were frozen at –18 °C and thawed after 22 weeks for the production of cooked ham. No significant difference was observed regarding physicochemical, color and microbiological parameters or in sensory acceptance. The hardness and chewiness parameters showed significant differences when compared to the control sample (ham made from chilled meat). Light microscopy of cooked ham samples showed that changes in the tissues were caused by freezing and thawing the meat. The effect of exudate was significant on the sodium content and compression force parameters, but this difference was not perceived in the sensory analysis, confirming that frozen pork meat can be used to produce cooked ham without loss of quality.

Syntrophic relationships between algae and bacteria in a fresh-water stream and in culture /

Interactions between freshwater algae and bacteria were examined in a natural stream habitat and a laboratory model. Field observations provided circumstantial evidence, in statistical correlation for syntrophy between the microbial populations. This relation is probably subject to control by the temperature and pH of the aquatic environment. Several species of a pond community were isolated in axenic culture and tests were performed to determine the nature of mixed species interactions. Isolation procedures and field studies indicated that selected strains of Chlorella and Azotobacter were closely associated in their natural habitat. With the suspected controlling parameters, pH and temperature, held constant, mixed cultures of algae and bacteria were compared to axenic cultures of the same organisms, and a mutual stimulation of growth was observed. A mixed pure culture apparatus was designed in this laboratory to study the algal-bacterial interaction and to test the hypothesis that such an interaction may take place through a diffusable substance or through certain medium-borne conditions, Azotobacter was found to take up a Chlorella-produced exudate, to stimulate protein synthesis, to enhance chlorophyll production and to cause a numerical increase in the interacting Chlorella population. It is not clear whether control is at the environmental, cellular or genetic level in these mixed population interactions. Experimental observations in the model system, taken with field correlations allow one to state that there may be a direct relationship governing the population fluctuations of these two organisms in their natural stream surroundings.

L-Glutamate uptake, decarboxylation to -aminobutyric acid and GABA efflux in isolated Asparagus sprengeri mesophyll cells

Addition of L-glutamate caused alkalinization of the medium surrounding Asparagus spreng.ri mesophyll cells. This suggests a H+/L-glutmate symport uptake system for L-glutamate. However stoichiometries of H+/L-glutamate symport into Asparagus cells were much higher than those in other plant systems. Medium alkalinization may also result from a metabolic decarboxylation process. Since L-glutmate is decarboxylated to r-amino butyric acid (SABA) in this system, the origin of medium alkalinization was reconsidered. Suspensions of mechanically isolated and photosyntheically competent Asparagus sprengeri mesophyll cells were used to investigate the H+/L-glutamate symport system, SABA production, GABA transport, and the origin of L-glutamate dependent medium alkalinization. The major results obtained are summarized as follows: 1. L-Glutamate and GABA were the second or third most abundant amino acids in these cells. Cellular concentrations of L-glutamate were 1.09 mM and 1.31 mM in the light and dark, respectively. Those of SABA were 1.23 mM and 1.17 mM in the light and dark, respectively. 2. Asparagine was the most abundant amino acid in xylem sap and comprised 54 to 68 1. of the amino acid pool on a molar basis. GABA was the second most abundant amino acid and represented 10 to 11 1. of the amino acid pool. L-Slutamate was a minor component. 3. A 10 minute incubation with 1 mM L-glutamate increased the production of GABA in the medium by 2,743 7. and 2,241 7. in the light and dark, respectively. 4. L-Glutamate entered the cells prior to decarboxylation. 5. There was no evidence for a H+/GABA symport process • 6. GABA was produced by loss of carbon-1 of L-glutamate. 7. The specific activity of newly synthesized labeled GABA suggests that it is not equilibrated with a storage pool of GABA. 8. The mechanism of GABA efflux appears to be a passive process. 9. The evidence indicates that the origin of L-glutamate dependent medium alkalinization is a H+/L-glutamate symport not an extracellular decarboxylation. The possible role of GABA production in regulating cytoplasmic pH and L-glutamate levels during rapid electrogenic H+/L-glutamate symport is discussed.

Bacteriophages of Erwinia amylovora and their potential use in biological control

Forty-four bacteriophage isolates of Erwinia amy/ovora, the causal agent of fire blight, were collected from sites in and around the Niagara Region of Southern Ontario in the summer of 1998. Phages were isolated only from sites where fire blight was present. Thirty-seven of these phages were isolated from the soil surrounding infected trees, with the remainder isolated from aerial plant tissue samples. A mixture of six E. amy/ovora bacterial host strains was used to enrich field samples in order to avoid the selection bias of a single-host system. Molecular characterization of the phages with a combination of peR and restriction endonuclease digestions showed that six distinct phage types were isolated. Ten phage isolates related to the previously characterized E. amy/ovora phage PEa1 were isolated, with some divergence of molecular markers between phages isolated from different sites. The host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amy/ovora strains, and that some types were able to lyse the epiphytic bacterium Pantoea agg/omerans. Biological control of E. amy/ovora by the bacteriophages was assessed in a bioassay using discs of immature pear fruit. Twenty-three phage isolates were able to significantly suppress the incidence of bacterial exudate on the pear disc surface. Quantification of the bacterial population remaining on the disc surface indicated that population reductions of up to 97% were obtainable by phage treatment, but that elimination of bacteria from the surface was not possible with this model system.

Plant rhizosphere specificity and variability in the insect and plant adhesins, Madl and Mad2, within the genus Metarhizium suggest plant adaptation as an evolutionary force

Metarhizium is a soil-inhabiting fungus currently used as a biological control agent against various insect species, and research efforts are typically focused on its ability to kill insects. In section 1, we tested the hypothesis that species of Metarhizium are not randomly distributed in soils but show plant rhizosphere-specific associations. Results indicated an association of three Metarhizium species (Metarhizium robertsii, M. brunneum and M. guizhouense) with the rhizosphere of certain types of plant species. M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense, which was supported by in vitro experiments with grass root exudate. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types. In section 2, we explored the variation in the insect adhesin, Madl, and the plant adhesin, Mad2, in fourteen isolates of Metarhizium representing seven different species. Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions. Phylogenetic analysis of 5' EF-Ia, which is used for species identification, as well as Madl and Mad2 sequences demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1a than Madl. This suggests Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation. While other abiotic and biotic factors cannot be excluded in contributing to divergence, it appears that plant associations have been the driving factor causing divergence among Metarhizium species.

Einfluss exogener und endogener Faktoren auf den Gehalt sekundärer Pflanzenstoffe (Carotinoide und Polyphenole) von Möhre (Daucus carota L.) und Weizen (Triticum aestivum L.)

Die vielfältigen Funktionen der sekundären Pflanzenstoffe sowohl im Organismus der Pflanze, als auch im Körper des Menschen bieten der Wissenschaft ein weites Betätigungsfeld. Die Carotinoide findet man in fast allen Plastiden der Pflanze und sie erfüllen dort Aufgaben in Form von Pigmenten, Antioxidantien, Hormonen und zählen außerdem zu den wichtigsten Bestandteilen des Photosyntheseapparates. Im menschlichen Organismus hingegen wirken sie als Provitamin A und in den Endverästelungen der Blutgefäße bei niedrigem Sauerstoffpartialdruck. Des Weiteren besitzen sie die Fähigkeiten freie Radikale unschädlich zu machen und wirken in vitro als Fänger von energiereichem Singulettsauerstoff. Die Polyphenole, die man zu der Stoffgruppe der Phenole zählt, befinden sich in den Randschichten von Obst, Gemüse, Getreide und anderen Samen. Ihnen obliegt die Aufgabe die darunter befindlichen Gewebe vor antioxidativem Verderb zu schützen. Im Körper des Menschen dagegen besitzen sie eine gerinnungshemmende Wirkung, schützen die Zellen vor Oxidation und üben Fähigkeiten aus, die Krebs vorbeugen können. Im Zuge dieser Literaturarbeit werden endogene und exogene Faktoren beschrieben, die auf Pflanzen allgemein und auf die Fokusprodukte Möhre (Daucus carota L.) und Weizen (Triticum aestivum L.) speziell einwirken. Die pflanzenphysiologische Herkunft und Bedeutung der sekundären Pflanzenstoffgruppen Carotinoide und Polyphenole wird dargestellt. Schließlich wird die vorhandene Literatur ausgewertet, die sich mit der Beeinflussung des Gehaltes der genannten sekundären Pflanzenstoffe in den gewählten Fokusprodukten durch exogene und endogene Faktoren beschäftigt. Die Beeinflussung des Polyphenolgehaltes in Möhre und des Carotinoid- und Polyphenolgehaltes in Weizen ist nur wenig untersucht. Dagegen ist die Beeinflussung des Carotinoidgehaltes in Möhren durch exogene und endogene Faktoren gut beschrieben. Der Faktor „Sorte“ spielt aufgrund der vorhandenen genetischen Anlagen (carotinoidreich / carotinoidarm) eine wesentliche Rolle bei der späteren Ausbildung des Carotinoidgehaltes in der Möhre. Die Reife der Möhre, die u.a. das Ergebnis des Einwirkens exogener Faktoren, wie Temperatur, Wuchsraum, verfügbare Wassermenge im Boden sowie der Niederschläge ist, beeinflusst maßgeblich den späteren Gehalt an Carotinoiden. Des Weiteren üben noch anbautechnische Maßnahmen (z.B. Düngung, Herbizidbehandlungen, Produktionstechnik) einen Einfluss auf den Carotinoidgehalt der Möhre aus. Der Phenolgehalt in Möhren wurde bisher ausschließlich auf Sortenebene verglichen. In einer Studie von Zhang & Hamauzu (2004) fand man heraus, dass der Phenol-Gehalt in den verschiedenen Geweben der Möhre von der Schale in Richtung Phloem und Xylem anstieg, während sich die antioxidantischen und radical scavening Aktivitäten auf gleiche Weise, wie der Phenol-Gehalt erhöhten und wiederum mit dem totalen Phenol-Gehalt korrelierten. Die phenolischen Extrakte verfügten über stärkere radical scavening Fähigkeiten, als die zum Vergleich herangezogenen Reinsubstanzen Chlorogensäure, Vitamin C und β-Carotin. Insgesamt wurde aufgrund dieser Studie vermutet, dass sich der höchste Gehalt an Phenolen in der Schale der Möhre befindet. Das geringe Vorliegen von Studien bezüglich des Carotinoid- und Phenolgehaltes in Weizen kann man darauf zurückführen, dass die sekundären Pflanzenstoffe im Vergleich zum Proteingehalt keine wesentliche Rolle als Qualitätsmerkmal beim Fokusprodukt Weizen spielen. Der Gehalt an Phenolen und Carotinoiden wurde bisher ausschließlich auf Sortenebene untersucht. Die Untersuchungen ergaben, dass der Gehalt an sekundären Pflanzenstoffen (Phenole, Tocopherole, Carotinoide) stark durch die Sorte beeinflusst wird.

Les sHsps en surera: capacitat protectora enfront l'estrés i variabilitat genètica

Els organismes responen a la temperatura i a molts altres estressos sintetitzant un grup de proteïnes anomenat proteïnes de xoc de calor (HSPs). En plantes les sHsps, d'entre 15 i 30 kDa formen el grup més abundant i divers, classificat en funció de la seva localització subcel.lular i homologia en: mitocondrials, cloroplàstiques, de reticle endoplasmàtic i citoplàsmiques de classe I i II. Les sHsps-CI s'ha descrit que s'indueixen per estrès tèrmic, hídric i oxidatiu (peròxid d'hidrògen, llum UV, ozó) i en resposta a algunes hormones. També s'expressen durant el desenvolupament, per exemple durant l'embriogènesi, on es creu que podrien tenir un paper protector de l'embrió enfront la dessecació. Tot i que hi ha abundants treballs que correlacionen la resistència a l'estrès i l'acumulació de sHsps-CI, els mecanismes moleculars d'aquesta activitat són poc conguts. Tot i això, per diverses sHsps-CI ha estat descrita una activitat xaperona in vitro i, més recentment, que la seva sobreexpressió augmenta la viabilitat de cèl.lules d'E.coli en condicions d'estrès tèrmic. L'estudi de l'acumulació de sHsps-CI en surera (Quercus suber) mitjançant immunodetecció en electroforesi bidimensional mostra uns patrons d'acumulació complexos i formats per dos grups d'espècies proteiques principals, a l'entorn dels 10 i 17 kDa respectivament, que mostren una inducció diferencial en funció del teixit i l'estrès. Mentre que les espècies proteiques de 17 kDa s'indueixen per temperatura però no per estrès oxidatiu, les de ca. 10 kDa ho fan per estrès oxidatiu i no per temperatura. Ambdós grups d'espècies proteiques s'acumulen conjuntament en fel.lema. Assajos de PCR i RT-PCR han permès clonar parcialment tres noves sHsps-CI en surera: Qshsp10-CI, QshspC-CI i QshspD-CI. Aquest fet confirma la multigeneïcitat de les sHsps-CI en surera que apuntava el patró bidimensional. Dels nous clons obtinguts destaca especialment Qshsp10-CI, un gen que presenta un codó stop enmig del domini -cristal.lí que fa que a la proteïna que se'n dedueix li manqui un 55% del domini -cristal.lí i tota l'extensió C-terminal. Es tractaria de la sHsp més petita i més truncada descrita fins al moment. L'anàlisi de l'expressió de Qshsp10-CI mitjançant RT-PCR mostra expressió en plantes tractades amb H2O2 però no en les que han estat sotmeses a un xoc de calor. Aprofitant l'oportunitat que oferia aquesta sHsp-CI de ser utilitzada com a model per l'estudi de la importància del domini -cristal.lí i l'extensió C-terminal en l'activitat protectora enfront l'estrès, es va voler determinar la capacitat que tenia d'augmentar la viabilitat de cèl.lules d'E. coli en condicions d'estrès tèrmic i oxidatiu. Els resultats mostren que la proteïna recombinant QsHsp10-CI, tot i la important truncació que té, és capaç de protegir cèl.lules d'E. coli en condicions d'estrès tèrmic i, remarcablement, en condicions d'estrès oxidatiu. Tots aquests resultats indiquen que les espècies proteiques de ca. 10 kDa podrien correspondre a Qshsp10-CI i tenir un paper en les cèl.lules del fel.lema en la protecció enfront l'estrès oxidatiu. L'estrès oxidatiu provoca lesions al DNA que poden produir errors en la replicació, transcripció o traducció i generar proteïnes aberrants. Donades les condicions d'estrès oxidatiu a les quals es troben sotmeses les cèl.lules del fel.lema, s'ha volgut estudiar la variabilitat dels seus àcids nucleics. La determinació de la taxa de mutació de la regió codificant del gen Qshsp17.4-CI en mRNA i DNA de fel.lema i àpex radicular, un teixit jove i en creixement actiu va mostrar unes taxes sorprenentment elevades en l'mRNA (1/1784 pb) i el DNA genòmic (1/1520 pb) del fel.lema. Aquestes taxes són les més altes descrites en un genoma nuclear eucariota i són similars a les dels virus d'RNA d'evolució ràpida com el virus de l'Hepatitis C. Amb aquestes taxes de mutació, un terç dels mRNAs del fel.lema de la surera contindrien missatges aberrants i la supervivència de les cel.lules es veuria compromesa. Això implica que el fel.lema hauria de ser considerat com un mosaic de cèl.lules genèticament heterogènies i, per tant, una sola seqüència no defineix en tota la seva amplitud un gen en aquest teixit. No es va detectar cap mutació en àpex de rel. Amb l'objectiu d'aprofundir en el coneixement de les mutacions que es donen en aquests dos teixits i per tal de poder fer una anàlisi qualitativa més completa que permetés especular sobre el seu origen, es va aplicar un mètode de selecció de seqüències mutants en base a la utilització d'enzims de restricció. Les mutacions detectades en fel.lema es corresponen amb les relacionades, en altres sistemes no nuclears (plasmidis, fags i DNA bacterià), amb l'estrès oxidatiu. En conseqüència, l'estrès oxidatiu al qual estan sotmeses les cèl.lules del fel.lema podria ser el causant de l'elevada taxa de mutació detectada. D'acord amb això, el tipus majoritari de productes d'oxidació de les bases del DNA que s'acumulen en brots de plàntules de surera en resposta al peròxid d'hidrògen produeixen el mateix tipus de mutacions detectades en l'mRNA del fel.lema de la surera. La major sensibilitat d'aquest nou mètode ha permès, a més, detectar mutacions en molècules d'mRNA de rel, un teixit en el qual no s'havia trobat cap mutació utilitzant el mètode de clonatge i seqüenciació directa. Tot i això, el tipus de mutacions predominants no estan relacionades amb l'estrès oxidatiu sinó amb erros en la reparació dels àcids nucleics.

Uneven distribution of nutrients in the root zone affects the incidence of blossom end rot and concentration of calcium and potassium in fruits of tomato

Tomato plants ( Lycopersicon esculentum Mill. var. DRK) were grown hydroponically to determine the effect of an uneven distribution of nutrients in the root zone on blossomend rot (BER) and Ca and K concentrations in the fruits. The plants were grown in rockwool with their root system divided into two portions. Each portion was irrigated with nutrient solutions with either the same or the different electrical conductivity (EC) in the range 0 to 6 dS m(-1). Solutions with high EC supplied to both sides of the root system significantly increased the incidence of BER. However, when only water or a solution of low EC was supplied to one portion, BER was reduced by 80%. Fruit yields were significantly higher ( P < 0.01) for plants that received solutions of the uneven EC treatments (6/0 or 4.5/0 EC treatment). Plants supplied with solutions of uneven EC generally had higher leaf and fruit concentrations of Ca but lower concentrations of K than those supplied with solutions of high EC. There was no difference in Ca concentration at the distal end of young fruits of the uneven EC treatment but it was reduced in the high EC treatments. The concentration of K in the mature fruits of the uneven EC treatments was lower than that of the high EC treatments and higher or similar that of the 3/3 or 2.5/2.5 EC treatments ( controls). A clear relationship was found between the incidence of BER and the exudation rate. High rate of xylem exudation was observed in the uneven EC treatments. Reduction of BER in the uneven EC treatments is most likely to be the effect of high exudation rate on Ca status in the young fruits. It was concluded that high EC of solution had positive effects on Ca concentration and incidence of BER provided that nutrient solution with low EC or water is supplied to the one portion of the root system.

Use of an artificial root to examine the influence of 8-hydroxyquinoline on soil microbial activity and bacterial community structure

Invasive plant species have been shown to alter the microbial community composition of the soils they invade and it is suggested that this below-ground perturbation of potential pathogens, decomposers or symbionts may feedback positively to allow invasive success. Whether these perturbations are mediated through specific components of root exudation are not understood. We focussed on 8-hydroxyquinoline, a putative allelochemical of Centaurea diffusa (diffuse knapweed) and used an artificial root system to differentiate the effects of 8-hydroxyquinoline against a background of total rhizodeposition as mimicked through supply of a synthetic exudate solution. In soil proximal (0-10 cm) to the artificial root, synthetic exudates had a highly significant (P < 0.001) influence on dehydrogenase, fluorescein diacetate hydrolysis and urease activity. in addition, 8-hydroxyquinoline was significant (p = 0.003) as a main effect on dehydrogenase activity and interacted with synthetic exudates to affect urease activity (p = 0.09). Hierarchical cluster analysis of 16S rDNA-based DGGE band patterns also identified a primary affect of synthetic exudates and a secondary affect of 8-hydroxyquinoline on bacterial community structure. Thus, we show that the artificial rhizosphere produced by the synthetic exudates was the predominant effect, but, that the influence of the 8-hydroxyquinoline signal on the activity and structure of soil microbial communities could also be detected. (C) 2009 Elsevier Ltd. All rights reserved.

Enhancement of germination of spores of Verticillium dahliae and Fusarium oxysporum esp vasinfectum in vascular fluid from cotton plants infected with the root-knot nematode

Root-knot nematode [RKN] (Meloidogyne incognita) can increase the severity of Verticillium (V dahliae) and Fusarium (F oxysporum f.sp. vasinfectum) wilt diseases in cotton (Gossypium hirsutum). This study was conducted to determine some of the physiological responses caused by nematode invasion that might decrease resistance to vascular wilt diseases. The effect of RKN was investigated on spore germination and protein, carbohydrate and peroxidase content in the xylem fluids extracted from nematode-infected plants. Two cotton cultivars were used with different levels of resistance to both of the wilt pathogens. Spore germination was greater in the xylem fluids from nematode-infected plants than from nematode-free plants. The effect on spore germination was greater in the Fusarium-resistant cultivar (51%). Analysis of these fluids showed a decrease in total protein and carbohydrate levels for both wilt-resistant cultivars, and an increase in peroxidase concentration. Fluids from nematode-free plants of the Verticillium-resistant cultivar contained 46% more peroxidase than the Fusarium-resistant cultivar. The results provide further evidence that the effect of RKN on vascular wilt resistance is systemic and not only local. Changes in metabolites in the xylem pass from the root to the stem, accelerating disease development.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL), for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis. Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification / detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL capture is at least twice that of control, untreated sample.

Juvenile-mature wood transition in pine: correlation between wood properties and candidate gene expression profiles

There is a strong desire to exploit transcriptomics data from model species for the genetic improvement of non-model crops. Here, we use gene expression profiles from the commercial model Pinus taeda to identify candidate genes implicated in juvenile-mature wood transition in the non-model relative, P. sylvestris. Re-analysis of 'public domain' SAGE data from xylem tissues of P. taeda revealed 283 mature-abundant and 396 juvenile-abundant tags (P < 0.01), of which 70 and 137, respectively matched to genes with known function. Based on sequence similarity, we then isolated 16 putative homologues of genes that in P. taeda exhibited widest divergence in expression between juvenile and mature samples. Candidate expression levels in P. sylvestris were almost invariably differential between juvenile and mature woody tissue samples among two cohorts of five trees collected from the same seed source and selected for genetic uniformity by genetic distance analysis. However, the direction of differential expression was not always consistent with that described in the original P. taeda SAGE data. Correlation was observed between gene expression and juvenile-mature wood anatomical characteristics by OPLS analysis. Four candidates (alpha-tubulin, porin MIP1, lipid transfer protein and aquaporin like protein) apparently had greatest influence on the wood traits measured. Speculative function of these genes in relation to juvenile-mature wood transition is briefly explored. Thus, we demonstrate the feasibility of exploiting SAGE data from a model species to identify consistently differentially expressed candidates in a related non-model species.

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

Shoot dieback in clipped young Golden Leyland (Cupressocyparis leylandii) trees - a physiological mechanism?

Shoot dieback is a problem in frequently trimmed Leyland hedges and is increasingly affecting gardeners’ choice of hedge trees, having a negative effect on a conifer nursery industry. Some damage can be attributed to the feeding by aphids, but it is unclear if there are also underlying physiological causes. In this study, we tested the hypothesis that shoot-clipping of conifer trees during adverse growing conditions (i.e. high air temperature and low soil moisture) could be leading to shoot ‘dieback’. Three-year-old Golden Leyland Cypress (x Cupressocyparis leylandii ‘Excalibur Gold’) plants were subjected to either a well-watered or droughted irrigation regime and placed in either a ‘hot’ (average day temperature = 40°C) or a ‘cool’ (average day temperature = 27°C) glasshouse compartment. Half of the plants from each glasshouse were clipped on Day 14 and again on Day 50. Measurements of soil moisture content (SMC), net CO2 assimilation rate (A), stomatal conductance (gs), branchlet xylem water potential (XWP), plant height and foliage colour were made. Within the clipped and unclipped treatments of both glasshouse compartments, plants from the droughted regime had significantly lower values for A, gs and XWP than those from the well-watered regime. However, there was no difference in these parameters between the hot and cool glasshouse compartments. The trends seen for A, gs and XWP of all treatments generally mirrored changes in SMC indicating a direct effect of water supply on these parameters. By the end of the experiment the overall foliage colour of plants from the hot glasshouse was darker than that of plants from the cool glasshouse and the overall foliage colour was also darker following shoot clipping. In general, shoot clipping led to increases in A, gs XWP and SMC. This may be due to the reduction in total leaf area leading to a greater supply of water for the remaining leaves. No shoot ‘dieback’ was observed in any treatment in response to drought stress or shoot-clipping.