Increases of secondary metabolite production in various plant cell cultures by co-cultivation with cork

Cork tissues increased secondary metabolite production of various plant cell cultures in a different manner from those of conventional elicitors. In Sophora flavescens and Glycyrrhiza glabra cultured cells, cork tissues increased the amounts of both lipophilic and hydrophilic flavonoids without affecting the cell growth, although elicitors such as copper ion and yeast extracts showed a clear inhibition of cell growth with the increasing amount of these lipophilic ones. The validity of this effect of cork tissues covered a wide range of aromatic compounds produced by suspension cell cultures derived from diverse plant species. Woody tissues of Japanese cypress had a very similar effect to that of cork. Partial purification of cork tissues suggested that the production-stimulating factor was present in the hemicellulose B fraction that was not included in the dedifferentiated cultured tissues.     

FhuA barrel-cork hybrids are active transporters and receptors

The crystal structure of Escherichia coli FhuA reveals a beta-barrel domain that is closed by a globular cork domain. It has been assumed that the proton motive force of the cytoplasmic membrane through the interaction of the TonB protein with the TonB box of the cork opens the FhuA channel. Yet, deletion of the cork results in an FhuA derivative, FhuADelta5-160, that still displays TonB-dependent substrate transport and phage receptor activity. To investigate this unexpected finding further, we constructed FhuADelta5-160 derivatives of FhuA proteins from Salmonella paratyphi B, Salmonella enterica serovar Typhimurium, and Pantoea agglomerans. The FhuADelta5-160 proteins inserted correctly into the outer membrane, and with the exception of the P. agglomerans protein, transported ferrichrome and albomycin. FhuA hybrids consisting of the beta-barrel of one strain and the cork of another strain were active and showed higher TonB-dependent ferrichrome transport rates than the corkless derivatives. Exceptions were the E. coli beta-barrel/Salmonella serovar Typhimurium cork hybrid protein and the Salmonella serovar Typhimurium beta-barrel/P. agglomerans cork hybrid protein, both of which were less active than the beta-barrels alone. Each of the FhuA mutant proteins displayed activity for each of their ligands, except for phage T5, only when coupled to TonB. The hybrid FhuA proteins displayed a similar activity with the E. coli TonB protein as with their cognate TonB proteins. Sensitivity to phages T1, T5, and phi80, rifamycin CGP 4832, and colicin M was determined by the beta-barrel, whereas sensitivity to phage ES18 and microcin J25 required both the beta-barrel and cork domains. These results demonstrate that the beta-barrel domain of FhuA confers activity and specificity and responds to TonB and that the cork domains of various FhuA proteins can be interchanged and contribute to the activities of the FhuA hybrids.     

Effect of fungal colonization on mechanical performance of cork

The industrialization of traditional processes relies on the scientific ability to understand the empirical evidence associated with traditional knowledge. Cork manufacturing includes one operation known as stabilization, where humid cork slabs are extensively colonized by fungi. The implications of fungal growth on the chemical quality of cork through the analysis of putative fungal metabolites have already been investigated. However, the effect of fungal growth on the mechanical properties of cork remains unexplored. This study investigated the effect of cork colonization on the integrity of the cork cell walls and their mechanical performance. Fungal colonization of cork by Chrysonilia sitophila, Mucor plumbeus Penicillium glabrum, P. olsonii, and Trichoderma longibrachiatum was investigated by microscopy. Growth occurred primarily on the surface of the cork pieces, but mycelium extended deeper into the cork layers, mostly via lenticular channels and by hyphal penetration of the cork cell wall.In this first report on cork decay in which specific correlation between fungal colonization and mechanical proprieties of the cork has been investigated, all colonizing fungi except C. sitophila, reduced cork strength, markedly altering its viscoelastic behaviour and reducing its Young's modulus.     

Cork stoppers industry: defining appropriate mould colonization

AIMS: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. METHODS AND RESULTS: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. CONCLUSIONS: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.     

A new method for measurement of annual growth rings in cork by means of autofluorescence

A new method for measuring annual growth rings in cork has been developed. It is based on the fact that excitation of Quercus suber L. cork cross-sectional planes with UV light and also with blue light results in enhanced autofluorescence at the growth ring borders. This distinct autofluorescence band is located in the cork produced at the end of the vegetation period, with its smaller cells and thicker cell walls. The enhanced autofluorescence of polyphenolics mainly results from a very thick suberin layer in the walls of the small late cork cells. The gradient in autofluorescence from late cork to first early cork is steep. The best visibility of cork annual rings was found under the epifluorescence microscope in 60 μm thick microtome cork cross sections. For fast screening of high sample numbers scanning the blue-excited (473 nm) fluorescence of water wetted polished cross-sectional surfaces of cork pieces with a laser-equipped fluorescence image analyzer was most suitable. Evaluation of visibility of cork rings showed a clear improvement in comparison with reflected light image analysis; thus data obtained with this new autofluorescence scanning method are excellent for modeling the yearly growth increment of cork.     

两种类型栓皮栎软木细胞微观排列与形态特征

采用扫描电镜对秦岭北坡楼观台地区的厚皮、薄皮两种类型栓皮栎软木进行细胞微观构造观察分析,并与欧洲栓皮槠进行比较,以阐明厚皮、薄皮栓皮栎软木的相关特性,为中国栓皮栎软木的合理利用提供依据。结果表明:(1)两种类型栓皮栎软木细胞的排列结构较一致,均由内部中空的封闭型薄壁细胞紧密排列组成;在弦切面上呈蜂窝状排列,径切面和横切面上呈砖墙状排列;在径切面上,软木细胞侧高整齐地排列成行,且与树干轴向垂直;在横切面上,软木细胞侧高整齐地处于以树干轴为中心散发出来的射线上。(2)栓皮栎软木细胞大小、细胞壁和侧壁褶皱等受生长季节的影响;从软木细胞形态特征上看,厚皮类型软木细胞壁薄、细胞体积大,其软木质量优于薄皮类型。(3)与欧洲栓皮槠比较,发现厚皮类型栓皮栎早软木细胞棱柱高较小(20.6μm vs.(对比)30~40μm),软木细胞壁略厚(1.7μm vs.1~1.5μm),细胞实体积(细胞壁体积占细胞总体积比例)略大(18.75%vs.10%),厚皮类型栓皮栎软木比欧洲栓皮槠的软木质量差一些。(4)受树皮生长应力的影响,两种类型栓皮栎软木细胞侧高壁上多发生褶皱,早软木细胞褶皱严重,晚软木细胞没有褶皱,但在早晚软木交界或含有杂质处褶皱特别严重,表明厚皮类型软木细胞的侧壁褶皱程度高于薄皮类型。(5)对细胞形态特征及软木特性等的分析表明,薄皮类型栓皮栎软木质量比厚皮类型差,未来对软木资源的开发利用应更注重厚皮类型。     

Analysis of raw cork production in Portugal and Catalonia using life cycle assessment

Purpose

This study aims to (1) evaluate the environmental impacts associated with the three types of raw cork produced in Portuguese cork oak woodlands (in Alentejo region) considering two alternative practices for stand establishment (plantation and natural regeneration), (2) compare the environmental impacts of raw cork production in Portuguese cork oak woodlands and in Catalonian cork oak forests, and (3) assess the influence of different allocation criteria for partitioning the environmental impacts between the different types of raw cork produced.

Methods

A cradle-to-gate approach was adopted starting with stand establishment up to cork storage in a field yard. The system boundaries include all management operations undertaken during the following stages: stand establishment, stand tending, cork stripping, and field recovery. The allocation of the environmental impacts to reproduction, second, and virgin cork was based on mass and market price criteria. An alternative allocation approach was simulated by allocating environmental impacts also to the wood produced in the cork oak stands. The impact assessment was performed using the characterization factors recommended by the International Reference Life Cycle Data System (ILCD).

Results and discussion

In Portugal, cork produced from naturally regenerated stands has a better environmental performance than cork produced from planted stands, but the differences are smaller than 10 %. Different management models of cork oak stands in Portugal and Catalonia (agro-silvopastoral system and forest system, respectively) originate different impact levels, which tend to be significantly lower in Catalonia. The environmental hot spots in the two regions are also distinct. In Catalonia, they are associated with cleaning, road maintenance, and worker and cork transport. In Portugal, they are fertilization, pruning, and cleaning. The two allocation criteria affect significantly the results obtained for virgin cork in Portugal and for virgin and second cork in Catalonia. Besides, when impacts are also allocated to wood, mass allocation should be avoided as it would not create incentives for a sustainable management of cork oak stands.

Conclusions

The environmental impact from Catalonian cork may be reduced by decreasing mechanized shrub cleaning and road maintenance operations through the introduction of livestock in cork oak forests, and also by a better planning of management operations. For the Portuguese cork, improvements may be achieved by optimizing fertilizer dosage, planting nitrogen-fixing crops and pastures that improve soil quality, avoiding unnecessary operations, improving the efficiency of management operations, and increasing tree density.     

In vivo reconstitution of the FhuA transport protein of Escherichia coli K-12

The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and phi80 and for colicin M and microcin J25. The crystal structure reveals a beta-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a beta-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the beta-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type beta-barrel to form an active FhuA. Previous claims that the beta-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the beta-barrel, and they suggest that in wild-type FhuA the beta-barrel is formed prior to the insertion of the cork.     

Climate response of cork growth in the Mediterranean oak (Quercus suber L.) woodlands of southwestern Portugal

In the Mediterranean climate regions, drought events are expected to affect the growth of forests ecosystems by changing trees growth rates and eventually inducing shifts in their growth patterns. Cork oak (Quercus suber L.) is a strictly western Mediterranean tree species periodically harvested for its bark, the cork. So far, cork oak has received limited attention for dendroclimatological studies due to its typical faint and erratic tree wood rings. Moreover, its distinct cork rings chronologies have been completely neglected. In this study we introduce an approach using cork ring chronologies dated back 9–10 years for climate response. Despite enhancing interannual variability and increasing statistical response to short-term climatic variability, still poorly understood, this study will possibly allow infer long-term climate response. We analyzed the cork ring chronologies of 55 cork samples collected in mature (under exploitation) trees in three distinct locations in southwestern Portugal. Cork growth recorded a high climate signal, with highly significant and coherent responses to the yearly climate-related sources of variation. We successfully assessed trends of cork growth via correlation analysis including selected climate variables among mean monthly temperature, monthly precipitation and, on an annual basis, eight precipitation indices. The high mean sensitivities and inter-series correlations found for cork ring chronologies combined with the significant variance explained by climate variables suggest that climate is likely one dominant signal that affects cork growth, but local environmental stresses can decisively affect this (climate) signal. Assuming cork growth as a proxy for cork oak growth, it seems conceivable that despite the trees being highly resistant to drought stress, cork oak woodlands in southwestern Portugal would have to face lesser growth in a global warming scenario.     

The Effect of Cork Pieces on Pseudohypericin Production in Cells ofHypericum perforatumShoots

The stimulating effect of cork pieces on hypericin and pseudohypericin biosyntheses was studied in cells of shoots regenerated from the callus cultures of St. John's wort (Hypericum perforatumL.). The addition of the cork matrix slightly stimulated shoot growth and enhanced pseudohypericin biosynthesis about threefold (to 0.4 mg/g dry wt). Pseudohypericin production increased proportionally with the amount of cork material added (from 1 to 4 mg/ml of growth medium). Further increase in the amount of cork pieces inhibited both pseudohypericin production and shoot growth. Organic and aqueous extracts of cork pieces did not affect the production of these substances.     

Mycobiota in Portuguese 'normal' and 'green' cork throughout the manufacturing process of stoppers

The compounds responsible for the so-called 'cork taint' include, among others, some microbial metabolites which can be produced by the microbial population colonizing the unprocessed cork and stoppers. This study was intended to obtain information on the mycobiota associated with Portuguese cork throughout the manufacturing process of stoppers. Samples of barks and stoppers of both 'normal' and 'green' cork were examined. Moulds were isolated from 'normal' and 'green' cork throughout the entire cork stopper manufacturing process. Yeasts were rarely detected in the corks. Fungal contamination was not detected in finished stoppers from the company under study.     

美登木根的显微结构及其内生真菌的分布

本文采用石蜡永久制片和光学显微摄像的方法对美登木(Maytenus confertiflorus)根的显微结构及其内生真菌的分布进行了研究。结果表明, 美登木根的次生结构由周皮和维管组织构成; 周皮由木栓层、木栓形成层和栓内层组成, 其中木栓层由5～6 列长形细胞组成; 维管组织中次生韧皮部所占根径的比例达46%, 其薄壁细胞中内含物较丰富, 次生木质部中分布有导管和木射线及少量木薄壁组织; 在美登木木栓层和次生韧皮部中分布有菌丝片段、膨大的菌丝、菌丝团及分生孢子; 内生真菌只在一定区域的皮层和次生韧皮部细胞中分布。     

Interxylary cork of <Emphasis Type="Italic">Saussurea discolor</Emphasis> and <Emphasis Type="Italic">S. pygmaea</Emphasis> (Asteraceae)

The root anatomy of the subalpine to alpine plant species Saussurea discolor (Willd.) DC., and Saussurea pygmaea (Jacq.) Spreng., (Asteraceae) has been investigated by means of light and fluorescence microscopy on specimens of Austrian provenance. Both species develop a so called interxylary cork which mediates the splitting of the root into various strands. This phenomenon takes place in the secondary xylem and involves the development of a periderm separating the originally solid xylem cylinder. Interxylary cork is currently known from approximately 40 species of the Dicotyledones. This is the first report of this specific anatomical structure from the two studied species.     

Cork and cork products

At least $ 20,000,000 worth of cork products are annually manufactured in the United States, and efforts are being made by annual plantings of cork oaks in 25 States eventually to relieve American industry of its present total dependence upon foreign sources to supply the cork for this production.     

美登木根的显微结构及其内生真菌的分布

本文采用石蜡永久制片和光学显微摄像的方法对美登木（Maytenus confertiflorus）根的显微结构及其内生真菌的分布进行了研究.结果表明,美登木根的次生结构由周皮和维管组织构成;周皮由木栓层、木栓形成层和栓内层组成,其中木栓层由5～6列长形细胞组成;维管组织中次生韧皮部所占根径的比例达46%,其薄壁细胞中内含物较丰富,次生木质部中分布有导管和木射线及少量木薄壁组织;在美登木木栓层和次生韧皮部中分布有菌丝片段、膨大的菌丝、菌丝团及分生孢子;内生真菌只在一定区域的皮层和次生韧皮部细胞中分布.     

Stimulating the production of homoisoflavonoids in cell suspension cultures of Caesalpinia pulcherrima using cork tissue

It has previously been demonstrated that cork tissue increases the efficiency of the production of lipophilic secondary metabolites in diverse plant cell suspension cultures. In the present study, three new homoisoflavonoids--named dihydrobonducellin, 2'-methoxydihydrobonducellin, and 2'-methoxybonducellin--and bonducellin and isobonducellin were isolated from Caesalpinia pulcherrima cultured cells coincubated with cork tissue. Cork tissue increased the production of 2'-methoxybonducellin by about 7-fold relative to control cells, and more than 80% of the product was recoverable from the cork tissue. When cork tissue and methyl jasmonate or yeast extract were added simultaneously to the medium, the amount of 2'-methoxybonducellin produced increased further. The production of the other four homoisoflavonoids was enhanced by variable amounts. Our results indicate that the addition of cork tissue would be an effective technique for investigating formation of secondary metabolites that usually accumulate only in trace amounts.     

Siderophore transport through Escherichia coli outer membrane receptor FhuA with disulfide-tethered cork and barrel domains

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.