Biofiltration of airborne VOCs with green wall systems-Microbial and chemical dynamics.

Botanical air filtration is a promising technology for reducing indoor air contaminants, but the underlying mechanisms need better understanding. Here, we made a set of chamber fumigation experiments of up to 16 weeks of duration, to study the filtration efficiencies for seven volatile organic compounds (VOCs; decane, toluene, 2-ethylhexanol, α-pinene, octane, benzene, and xylene) and to monitor microbial dynamics in simulated green wall systems. Biofiltration functioned on sub-ppm VOC levels without concentration-dependence. Airflow through the growth medium was needed for efficient removal of chemically diverse VOCs, and the use of optimized commercial growth medium further improved the efficiency compared with soil and Leca granules. Experimental green wall simulations using these components were immediately effective, indicating that initial VOC removal was largely abiotic. Golden pothos plants had a small additional positive impact on VOC filtration and bacterial diversity in the green wall system. Proteobacteria dominated the microbiota of rhizosphere and irrigation water. Airborne VOCs shaped the microbial communities, enriching potential VOC-utilizing bacteria (especially Nevskiaceae and Patulibacteraceae) in the irrigation water, where much of the VOC degradation capacity of the biofiltration systems resided. These results clearly show the benefits of active air circulation and optimized growth media in modern green wall systems.

Morphological and molecular identification to secure cultivar maintenance and management of self-sterile Rubus arcticus.

BACKGROUND AND AIMS: Preservation of cultivar purity creates a particular challenge for plants that are self-incompatible, require insects for cross-pollination, and have easily germinating seeds and vigorously spreading rhizomes. As the fields must be planted with mixed populations, and a balance must be maintained between the cultivars to achieve effective pollination, methods for field monitoring of the relative density of different cultivars must be practical. Furthermore, a DNA-based method is needed for cultivar verification in the collections and outside of the growing season. The aim of this study was to develop both types of methods for Rubus arcticus (arctic bramble). METHODS: Morphological parameters were measured from six cultivars grown on three farms. Observations from the flowers and fruits included: petal and sepal number, flower diameter, arrangement of petals, size of calyx in relation to corolla, fruit weight, yield and soluble sugars. Observations from the leaves included: width and height of middle leaflet, shape of the base of terminal leaflet, shape of terminal leaflet, leaf margin serration and fingertip touch. The applicability of simple sequence repeat (SSR) or microsatellite DNA markers developed for red raspberry was tested on eight arctic bramble cultivars. KEY RESULTS AND CONCLUSIONS: Morphological and molecular identification methods were developed for R. arcticus. The best morphological characteristics were the length-to-width ratio of the middle leaflet and leaf margin serration. A particular characteristic, fingertip touch, was shown by electron microscopy to be related to the density and quality of the leaf hairs. Red raspberry SSR marker no. 126 proved to be applicable for differentiation of the eight arctic bramble cultivars tested. These identification methods are critical to secure the maintenance and management of R. arcticus. However, the challenges faced and approaches taken are equally applicable to other species with similar biology.

Plant metallothioneins--metal chelators with ROS scavenging activity?

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens.

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F(3) families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F(3) sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant Thlaspi caerulescens.

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.

Unintended effects and their detection in genetically modified crops.

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.

Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation for SvMT2b expression. Allele-specific PCR showed that low-expression F(3) plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent. SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F(3) plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

Flavonol content varies among black currant cultivars.

Flavonoids and related plant compounds in fruits and vegetables are of particular importance as they have been found to possess antioxidant and free radical scavenging activity. The HPLC-based quantitative procedure, with improved extraction and hydrolysis, was used to analyze the content of the flavonols quercetin, myricetin, and kaempferol in 10 black currant cultivars from organic farms and in 5 cultivars from conventional farms. Myricetin was the most abundant flavonol, and its amount varied significantly among cultivars, from 8.9 to 24.5 mg x 100 g(-1) (fresh weight). The quercetin levels in black currant also varied widely among the cultivars, from 5.2 to 12.2 mg x 100 g(-1). The kaempferol levels in black currant cultivars were low, ranging from 0.9 to 2.3 mg x 100 g(-1). The sum of these major flavonols varied widely among black currant cultivars. No consistent differences in the contents of flavonols were found between the same black currant cultivars grown in organic and conventional ways. The high variability in the levels of flavonols in different cultivars offers possible avenues for identifying and selecting cultivars rich in certain flavonols for the special production of berries for industrial use.

Clonal differences in copper and zinc tolerance of birch in metal-supplemented soils.

Metal tolerance of a range of birch clones (Betula pendula and Betula pubescens) originating from metal-contaminated sites in England, Wales, Belgium and Finland were tested in soils supplemented with several concentrations of copper (Cu) or zinc (Zn) (500, 2000, 5000 mg kg-1 dry wt. soil of CuSO(4).5H2O or ZnSO(4).7H2O) for 4 months and with sub-toxic metal supplements (500 mg CuSO4, 2000 mg ZnSO4) for 6 months. When grown at high concentrations of metals, severe toxicity symptoms (growth inhibition, chlorosis, necrosis) and clear evidence for differences in tolerance to this toxicity were found in a subset of the clones. When all clones were grown at a much lower, sub-toxic level of metal, again significant differences could be found between some of the clones. Clones derived from the same population varied greatly in their tolerance. However, the overall pattern of metal specificity varied in agreement with the type of soil contamination at the site of origin. The growth of the clones from Harjavalta Cu/nickel smelter area was 19% better in Cu than in Zn-supplemented soil, on average. The growth of clones from Maatheide Zn smelter are was 19% poorer in Cu- than in Zn-supplemented soil. Sensitive clones accumulated more Cu and Zn to the above-ground parts. Some birch clones were able to survive at about 20-fold higher than typical total background Cu or Zn concentrations, whereas most clones were able to grow without serious toxic symptoms at about 10-fold concentrations.

Influence of domestic processing and storage on flavonol contents in berries.

Effects of domestic processing and storage on the flavonols quercetin, myricetin, and kaempferol in five berries were studied using an optimized RP-HPLC method with UV and diode array detection after an acid hydrolysis of the corresponding glycosides. In fresh berries, the total content of flavonols was highest in lingonberry (169 mg/kg) and black currant (157 mg/kg), intermediate in bilberry (41 mg/kg) and strawberry (17 mg/kg), and lowest in red raspberry (9.5 mg/kg). Cooking strawberries with sugar to make jam resulted in minor losses (quercetin 15%, kaempferol 18%). During cooking of bilberries with water and sugar to make soup, 40% of quercetin was lost. Traditional preservation of crushed lingonberries in their own juice caused a considerable (40%) loss of quercetin. Only 15% of quercetin and 30% of myricetin present in unprocessed berries were retained in juices made by common domestic methods (steam-extracted black currant juice, unpasteurized lingonberry juice). Cold-pressing was superior to steam-extraction in extracting flavonols from black currants. During 9 months of storage at 20 C, quercetin content decreased markedly (40%) in bilberries and lingonberries, but not in black currants or red raspberries. Myricetin and kaempferol were more susceptible than quercetin to losses during storage.

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils.

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.

Content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries.

The amounts of quercetin, myricetin, and kaempferol aglycons in 25 edible berries were analyzed by an optimized RP-HPLC method with UV detection and identified with diode array and electrospray ionization mass spectrometry detection. Sixteen species of cultivated berries and nine species of wild berries were collected in Finland in 1997. Quercetin was found in all berries, the contents being highest in bog whortleberry (158 mg/kg, fresh weight), lingonberry (74 and 146 mg/kg), cranberry (83 and 121 mg/kg), chokeberry (89 mg/kg), sweet rowan (85 mg/kg), rowanberry (63 mg/kg), sea buckthorn berry (62 mg/kg), and crowberry (53 and 56 mg/kg). Amounts between 14 and 142 mg/kg of myricetin were detected in cranberry, black currant, crowberry, bog whortleberry, blueberries, and bilberry. Kaempferol was detected only in gooseberries (16 and 19 mg/kg) and strawberries (5 and 8 mg/kg). Total contents of these flavonols (100-263 mg/kg) in cranberry, bog whortleberry, lingonberry, black currant, and crowberry were higher than those in the commonly consumed fruits or vegetables, except for onion, kale, and broccoli.

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens.

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which ß-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation.

Induction of genes for the stress proteins PR-10 and PAL in relation to growth, visible injuries and stomatal conductance in birch (Betula pendula) clones exposed to ozone and/or drought.

Well watered (WW) or drought-stressed (DS) saplings of ozone-sensitive and ozone-tolerant (less sensitive) birch (Betula pendula Roth) clones were exposed for 43 d to 0 nl l-1 or 100 nl l-1 ozone. Relative growth rates of leaves, stem, and roots, leaf discolouration, stomatal conductance and induction of genes encoding stress-related proteins PR-10, PAL and a LEA-group protein BP8 were determined. In general, both ozone and drought stress, singly and in combination, increased transcript levels of PR-10 in both clones. This was related to lower induction of PAL (except in older leaves of the tolerant clone), and increased proportions of visibly injured and yellowed leaves in ozone-exposed plants. The clones differed in their stomatal conductance and growth responses. In the less sensitive clone 2, ozone did not affect growth rates, but high stomatal conductance was observed in WW ozone-exposed plants. The more sensitive clone 5 showed, on the contrary, reduced growth rates and low stomatal conductance in WW ozone plants. Interestingly, clone 2 was sensitive to drought stress alone, whereas clone 5 was highly sensitive to ozone and drought stress experienced together. The results show that appearance of visible injuries (necrotic flecks) and enhanced yellowing of leaves coincided with the induction of genes for stress proteins PR-10 and PAL. The short-term growth responses, however, seemed to be separate processes. Additionally, stomatal conductance was related to leaf injuries and growth rates in a complicated manner, emphasizing the complex nature of ozone sensitivity/tolerance mechanisms in birch.

Differential tolerance to copper and zinc of micropropagated birches tested in hydroponics.

Copper and zinc tolerances of 10 micropropagated birch (Betula pendula and B. pubescens) clones were studied in hydroponic culture. Tolerance indices were determined, based on the mean growth rate of the longest root in 1 wk. A seed-derived clone (142A), from a lead/Zn-contaminated site showed more tolerance to Cu and Zn than bud-derived clones (HA02 and HA18) from a Cu/nickel-contaminated site or an ozone-tolerant clone (KL-2-M) from an uncontaminated area. For Cu, the EC50 values were 30, 14, 8 and 11 µM in clones 142A, HA02, HA18 and KL-2-M, respectively. FOT Zn, the EC50 s were 4000 and 350 µM in clones 142A and KL-2-M, respectively. The relative Cu and Zn tolerances of the other clones were estimated by growing the plants in 30 µ CuSO4 , and in 2000 or 350 µM ZnSO4 , respectively. It is of interest that the Zn-tolerant clone 142A was tolerant to Cu, although this metal was present at a very low concentration in the soil where the parent tree grows. Another clone (142B), from another seed of the same parent tree, was tolerant neither to Zn nor Cu. Compared with their own EC20 s for root growth for Cu, 142A took up more Cu than KL-2-M, suggesting that the higher tolerance of the former clone is not explained by reduced uptake of Cu. The Zn uptake in clones 142A and KL-2-M was studied at 4000 µM and 800 µM Zn, respectively. Interestingly, the roots of both clones contained the same amount of Zn, even though clone 142A was exposed to a fivefold concentration of Zn.

Single-step immunocapture RT-PCR in the detection of raspberry bushy dwarf virus.

An immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) method for a highly sensitive analysis of raspberry bushy dwarf virus (RBDV) in infected plants is described. In the method, preliminary purification of virus particles or viral RNA from the plant material is not necessary. Viruses are enriched during the assay by antibodies bound in the PCR microplate wells, followed by lysis of the viral particles, and RT-PCR of the viral RNA. The reaction mixtures, including reverse transcriptase and DNA polymerase, have been selected so that both enzymes are active in the lysis and amplification conditions; by this way, it is possible to conduct the whole procedure in a single step. Using the method, four fragments from RNA-3 of RBDV have been amplified with various combinations of four primers. The procedure is sensitive enough to allow a simple detection of RBDV in in vitro cultured plants in which the detection of viruses by conventional immunological methods is difficult or even impossible.

P450 enzyme CYP2B catalyzes the detoxification of diisopropyl fluorophosphate.

Phenobarbital and some other enzyme-inducers are known to reduce organophosphate toxicity. One suggested mechanism is the induction of liver cytochrome P450 enzymes catalyzing monooxygenation reactions. The aim of the present study was to elucidate the cytochrome P450 subfamily, or P450 isoenzyme(s), participating in the detoxification of diisopropyl fluorophosphate (DFP) in the rat. DFP resulted in a type I spectrum in liver microsomes from phenobarbital- or RP 52028-treated rats (binding constants 0.32 and 0.17 microM, respectively) and in a purified P450 preparation enriched with CYP2B. The spectrum was reversible by metyrapone, an inhibitor of the CYP2B enzyme subfamily. The 7-pentoxyresorufin O-dealkylase activity was inhibited by DFP in liver microsomes from phenobarbital- or RP 52028-treated rats and in a reconstituted system containing the purified CYP2B preparation. In microsomes from phenobarbital-pretreated rats, the inhibition was of a mixed type, i.e., competitive-non-competitive (Km = 0.5 microM; Ki = 6 microM). The microsomal fractions of livers from phenobarbital- or RP 52028-treated rats detoxified DFP effectively in vitro, as measured by a decrease in the DFP inhibition of cholinesterase activity. This detoxification was antagonized by metyrapone and by an antibody raised against purified CYP2B preparation. Clotrimazole, an inhibitor of P450 enzymes, inhibited the detoxification of DFP in rat liver in vivo. A genetically-modified hamster cell line expressing CYP2B1 oxidized NADPH in the presence of DFP. No such oxidation was detected in the parent cell line. These studies suggest that CYP2B1 metabolizes DFP and may significantly contribute to the detoxification of this organophosphate in vivo.

Comparison of expression of aldehyde dehydrogenase 3 and CYP1A1 in dominant and recessive aryl hydrocarbon hydroxylase-deficient mutant mouse hepatoma cells.

The mouse hepatoma cell line Hepa-1 is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for both CYP1A1 (aryl hydrocarbon hydroxylase, AHH) and class 3 aldehyde dehydrogenase (ALDH3) enzymes. To test the hypothesis of a common regulatory mechanism, several AHH deficient mutants of Hepa-1 were studied for their ALDH3 activities and specific mRNA levels before and after TCDD treatment. The recessive (with respect to the wild-type Hepa-1) mutants have defects in Cypla-1 structural gene (mutant c1) or in the Ah (aryl hydrocarbon) receptor (mutants c2 and c6 with decreased levels of Ah receptor; mutant c4 defective in the DNA binding of the Ah receptor). The results with these mutants suggested that Ah receptor nuclear translocator protein, ARNT, is needed for ALDH3 expression. Two dominant mutants, one of which is characterized by preventing the binding of the Ah receptor complex to DNA, were also studied. Surprisingly, these mutants possessed elevated levels of ALDH3 mRNA and enzyme activities which were also inducible by TCDD. The binding of Ah receptor-ligand complex to DNA was thus not needed for the expression of ALDH3. A dominant repressor for Cypla-1 gene transcription did not prevent the derepression or induction of ALDH3. The results thus suggest that Aldh-3 gene is regulated by a mechanism independent of the Ah receptor.