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Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1 , PheCAD6 , PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2 , PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1 , PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.

Heat shock transcription factors (HSFs) are central elements in the regulatory network that controls plant heat stress response. They are involved in multiple transcriptional regulatory pathways and play important roles in heat stress signaling and responses to a variety of other stresses. We identified 41 members of the HSF gene family in moso bamboo, which were distributed non-uniformly across its 19 chromosomes. Phylogenetic analysis showed that the moso bamboo HSF genes could be divided into three major subfamilies; HSFs from the same subfamily shared relatively conserved gene structures and sequences and encoded similar amino acids. All HSF genes contained HSF signature domains. Subcellular localization prediction indicated that about 80% of the HSF proteins were located in the nucleus, consistent with the results of GO enrichment analysis. A large number of stress response-associated cis-regulatory elements were identified in the HSF upstream promoter sequences. Synteny analysis indicated that the HSFs in the moso bamboo genome had greater collinearity with those of rice and maize than with those of Arabidopsis and pepper. Numerous segmental duplicates were found in the moso bamboo HSF gene family. Transcriptome data indicated that the expression of a number of PeHsfs differed in response to exogenous gibberellin (GA) and naphthalene acetic acid (NAA). A number of HSF genes were highly expressed in the panicles and in young shoots, suggesting that they may have functions in reproductive growth and the early development of rapidly-growing shoots. This study provides fundamental information on members of the bamboo HSF gene family and lays a foundation for further study of their biological functions in the regulation of plant responses to adversity., (© 2021. The Author(s).)

Background: Moso bamboo, the fastest growing plant on earth, is an important source for income in large areas of Asia, mainly cultivated in China. Lateral organ boundaries domain (LBD) proteins, a family of transcription factors unique to plants, are involved in multiple transcriptional regulatory pathways and play important roles in lateral organ development, pathogen response, secondary growth, and hormone response. The LBD gene family has not previously been characterized in moso bamboo (Phyllostachys edulis)., Results: In this study, we identified 55 members of the LBD gene family from moso bamboo and found that they were distributed non-uniformly across its 18 chromosomes. Phylogenetic analysis showed that the moso bamboo LBD genes could be divided into two classes. LBDs from the same class share relatively conserved gene structures and sequences encoding similar amino acids. A large number of hormone response-associated cis-regulatory elements were identified in the LBD upstream promoter sequences. Synteny analysis indicated that LBDs in the moso bamboo genome showed greater collinearity with those of O. sativa (rice) and Zea mays (maize) than with those of Arabidopsis and Capsicum annuum (pepper). Numerous segmental duplicates were found in the moso bamboo LBD gene family. Gene expression profiles in four tissues showed that the LBD genes had different spatial expression patterns. qRT-PCR assays with the Short Time-series Expression Miner (STEM) temporal expression analysis demonstrated that six genes (PeLBD20, PeLBD29, PeLBD46, PeLBD10, PeLBD38, and PeLBD06) were consistently up-regulated during the rapid growth and development of bamboo shoots. In addition, 248 candidate target genes that function in a variety of pathways were identified based on consensus LBD binding motifs., Conclusions: In the current study, we identified 55 members of the moso bamboo transcription factor LBD and characterized for the first time. Based on the short-time sequence expression software and RNA-seq data, the PeLBD gene expression was analyzed. We also investigated the functional annotation of all PeLBDs, including PPI network, GO, and KEGG enrichment based on String database. These results provide a theoretical basis and candidate genes for studying the molecular breeding mechanism of rapid growth of moso bamboo.

Bamboo, a fast-growing non-timber forest plant with many uses, is a valuable species for green development. However, bamboo flowering is very infrequent, extending, in general, for up to 120 years. Ecologically, bamboo species are generally better adapted to various environments than other grasses. Therefore, the species deserves a special status in what could be called Ecological Bioeconomy. An understanding of the genetic processes of bamboo can help us sustainably develop and manage bamboo forests. Transposable elements (TEs), jumping genes or transposons, are major genetic elements in plant genomes. The rapid development of the bamboo reference genome, at the chromosome level, reveals that TEs occupy over 63.24% of the genome. This is higher than found in rice, Brachypodium, and sorghum. The bamboo genome contains diverse families of TEs, which play a significant role in bamboo's biological processes including growth and development. TEs provide important clues for understanding the evolution of the bamboo genome. In this chapter, we briefly describe the current status of research on TEs in the bamboo genome, their regulation, and transposition mechanisms. Perspectives for future research are also provided.

Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) is a rapidly growing grass of industrial and ecological importance. However, the molecular mechanisms of its remarkable growth are not well understood. In this study, we investigated the early-stage growth of moso bamboo shoots and defined three different growth stages based on histological and biochemical analyses, namely, starting of cell division (SD), rapid division (RD) and rapid elongation (RE). Further analyses on potentially relevant cellular pathways in these growth stages using multi-omics approaches such as transcriptomics and proteomics revealed the involvement of multiple cellular pathways, including DNA replication, repair and ribosome biogenesis. A total of 8045 differentially expressed genes (DEGs) and 1053 differentially expressed proteins (DEPs) were identified in our analyses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of detected DEGs identified several key biological pathways such as phytohormone metabolism, signal transduction, cell wall development and carbohydrate metabolism. The comparative analysis of proteins displayed that a total of 213 DEPs corresponded with DEGs and 3 significant expression profiles that could be promoting the fast growth of bamboo internodes. Moreover, protein-protein interaction network prediction analysis is suggestive of the involvement of five major proteins of signal transduction, DNA synthesis and RNA transcription, and may act as key elements responsible for the rapid shoot growth. Our work exploits multi-omics and bioinformatic approaches to unfurl the complexity of molecular networks involved in the rapid growth of moso bamboo and opens up questions related to the interactions between the functions played by individual molecular pathway., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dissolved organic matter (DOM) has an important effect on soil fertility, activity of microorganisms and transport of contaminants. In this study, DOM released by the hydrochar and biochar prepared under various conditions from pig manure, was assessed using a combination of UV-Visible spectroscopy, fluorescence excitation-emission (EEM) spectrophotometry and 1 H-nuclear magnetic resonance ( 1 H NMR). The dissolved organic carbon (DOC) extracted from the hydrochar and biochar ranged from 3.34-11.96% and 0.38-0.48%, respectively, and the highest DOM was released by HCK0.5 (180 °C and 0.5% KOH). The aliphatic compounds were most common in DOM which mainly included three humic acid-like and one protein-like substance. The hydrochar-DOM had a larger molecular weight and lower aromaticity than biochar-DOM, but the effect of temperature on the DOM characteristics of hydrochar and biochar was opposite. The acidic treatment increased the content of functional groups containing oxygen and nitrogen in hydrochar-DOM, and alkaline treatment increased the content of aliphatic compounds. The results obtained are beneficial to select carbonation process and guide the rational application of hydrochar and biochar from pig manure in soil remediation field., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Petroleum hydrocarbons (PHCs) continue to be among the most common pollutants in soil worldwide. Phytoremediation has become a sustainable way of dealing with PHC contamination. We conducted the off-site phytoremediation of PHC-polluted soil from an oil tanker truck accident, where poplars were used for the phytoremediation of the oil-polluted soil in a boreal climate during a seven-year treatment. The succession of bacterial communities over the entire phytoremediation process was monitored using microbial ecological tools relying on high-throughput 16S rRNA gene sequencing. Upon the successful depletion of PHCs from soil, endophytic communities were analyzed in order to assess the complete plant-associated microbiome after the ecological recovery. The rhizosphere-associated soil exhibited different bacterial dynamics than unplanted soil, but both soils experienced succession of bacteria over time, with diversity being negatively correlated with PHC concentration. In the relatively short growing season in North Europe, seasonal variations in environmental conditions were identified that contributed to the dynamics of bacterial communities. Overall, our study proved that phytoremediation using poplar trees can be used to assist in the removal of PHCs from soils in boreal climate conditions and provides new insight into the succession patterns of bacterial communities associated with these plants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2 , have previously been identified in moso bamboo ( Phyllostachys edulis ). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR's affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5-2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2 . The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

Northern peatlands in general have high methane (CH 4 ) emissions, but individual peatlands show considerable variation as CH 4 sources. Particularly in nutrient-poor peatlands, CH 4 production can be low and exceeded by carbon dioxide (CO 2 ) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO 2 to CH 4 produced. After [ 13 C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH 4 and CO 2 The oligotrophic fen had lower CH 4 production but produced 3 to 59 times more CO 2 than CH 4 RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH 4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria The oligotrophic peat with excess CO 2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH 4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO 2 production in peatlands., Importance: Peatlands are major sources of the greenhouse gas methane (CH 4 ), yet in many peatlands, CO 2 production from unresolved anaerobic processes exceeds CH 4 production. Anaerobic degradation produces the precursors of CH 4 production but also represents competing processes. We show that anaerobic degradation leading to high or low CH 4 production involved distinct sets of bacteria. Well-known fermenters dominated in a peatland with high CH 4 production, while novel and unconventional degraders could be identified in a site where CO 2 production greatly exceeds CH 4 production. Our results help identify and assign functions to uncharacterized bacteria that promote or inhibit CH 4 production and reveal bacteria potentially producing the excess CO 2 in acidic peat. This study contributes to understanding the microbiological basis for different levels of CH 4 emission from peatlands., (Copyright © 2017 American Society for Microbiology.)

Microorganisms capable of transforming toxic selenium oxyanions into non-toxic elemental selenium (Se°) may be considered as biocatalysts for the production of selenium nanoparticles (SeNPs), eventually exploitable in different biotechnological applications. Two Burkholderia fungorum strains (B. fungorum DBT1 and B. fungorum 95) were monitored during their growth for both capacity and efficiency of selenite (SeO 3 2- ) reduction and elemental selenium formation. Both strains are environmental isolates in origin: B. fungorum DBT1 was previously isolated from an oil refinery drainage, while B. fungorum 95 has been enriched from inner tissues of hybrid poplars grown in a soil contaminated by polycyclic aromatic hydrocarbons. Our results showed that B. fungorum DBT1 is able to reduce 0.5mM SeO 3 2- to Se° when cultured aerobically in liquid medium at 27°C, while B. fungorum 95 can reduce more than 1mM SeO 3 2- to Se° within 96h under the same growth conditions, with the appearance of SeNPs in cultures of both bacterial strains. Biogenic SeNPs were spherical, with pH-dependent charge and an average hydrodynamic diameter of 170nm and 200nm depending on whether they were produced by B. fungorum 95 or B. fungorum DBT1, respectively. Electron microscopy analyses evidenced that Se nanoparticles occurred intracellularly and extracellularly. The mechanism of SeNPs formation can be tentatively attributed to cytoplasmic enzymatic activation mediated by electron donors. Biogenic nanoparticles were then probably released outside the bacterial cells as a consequence of a secretory process or cell lysis. Nevertheless, formation of elemental selenium nanoparticles under aerobic conditions by B. fungorum DBT1 and B. fungorum 95 is likely due to intracellular reduction mechanisms. Biomedical and other high tech sectors might exploit these biogenic nanoparticles in the near future, once fully characterized and tested for their multiple properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Indigenous Cr(VI) reducing bacterial strains Pseudomonas aeruginosa Rb-1 and Ochrobactrum intermedium Rb-2 were evaluated for EPS production under Cr(VI) challenged and free conditions. Strain Rb-2 was more efficient in total EPS production (13.63 mg g(-1)) than Rb-1 (4.15 mg g(-1)) under Cr(VI) stress. Thick covering of capsular material around the cells of both bacterial strains was detected by electron microscopy. Transmission electron micrographs showed the appearance of pilli like structures under chromium stress by two bacteria suggested the possible involvement of this in exchange of hereditary material to increase their chances of survival under stress conditions. FTIR study showed involvement of sulphonate and hydroxyl groups in the binding with Cr(VI) ions. Solid-state (13) C NMR spectra revealed that EPS produced by both strains exhibited structural similarity with the glucan. The partial psl gene sequences of Rb-1 and Rb-2 showed homology with psl gene of Pseudomonas aeruginosa PAO1 and capsular polysaccharide biosynthesis protein of various strains of Pseudomonas. This is the first report on the identification of psl gene from Ochrobacterum in NCBI GenBank database up to our knowledge., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Poplars have widely been used for rhizoremediation of a broad range of organic contaminants for the past two decades. Still, there is a knowledge gap regarding the rhizosphere-associated bacterial communities of poplars and their dynamics during the remediation process. It is envisaged that a detailed understanding of rhizosphere-associated microbial populations will greatly contribute to a better design and implementation of rhizoremediation. To investigate the long-term succession of structural and catabolic bacterial communities in oil-polluted soil planted with hybrid poplar, we carried out a 2-year field study. Hybrid aspen (Populus tremula × Populus tremuloides) seedlings were planted in polluted soil excavated from an accidental oil-spill site. Vegetated and un-vegetated soil samples were collected for microbial community analyses at seven different time points during the course of 2 years and sampling time points were chosen to cover the seasonal variation in the boreal climate zone. Bacterial community structure was accessed by means of 16S rRNA gene amplicon pyrosequencing, whereas catabolic diversity was monitored by pyrosequencing of alkane hydroxylase and extradiol dioxygenase genes. We observed a clear succession of bacterial communities on both structural and functional levels from early to late-phase communities. Sphingomonas type extradiol dioxygenases and alkane hydroxylase homologs of Rhodococcus clearly dominated the early-phase communities. The high-dominance/low-diversity functional gene communities underwent a transition to low-dominance/high-diversity communities in the late phase. These results pointed towards increased catabolic capacities and a change from specialist to generalist strategy of bacterial communities during the course of secondary succession., (© 2014 John Wiley & Sons Ltd.)

Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We performed a grid-based sampling of an aged creosote-contaminated site followed by geostatistical modelling to illustrate the spatial patterns of microbial diversity and activity and to relate these patterns to the distribution of pollutants. Spatial distribution of bacterial groups unveiled patterns of niche differentiation regulated by patchy distribution of pollutants and an east-to-west pH gradient at the studied site. Proteobacteria clearly dominated in the hot spots of creosote pollution, whereas the abundance of Actinobacteria, TM7 and Planctomycetes was considerably reduced from the hot spots. The pH preferences of proteobacterial groups dominating in pollution could be recognized by examining the order and family-level responses. Acidobacterial classes came across as generalists in hydrocarbon pollution whose spatial distribution seemed to be regulated solely by the pH gradient. Although the community evenness decreased in the heavily polluted zones, basal respiration and fluorescein diacetate hydrolysis rates were higher, indicating the adaptation of specific indigenous microbial populations to hydrocarbon pollution. Combining the information from the kriged maps of microbial and soil chemistry data provided a comprehensive understanding of the long-term impacts of creosote pollution on the subsurface microbial communities. This study also highlighted the prospect of interpreting taxa-specific spatial patterns and applying them as indicators or proxies for monitoring polluted sites.

Chromium pollution is produced in connection with industrial processes like in tanneries. It has been suggested that bioremediation could be a good option for clean up. The stress effect of variable chromate levels, pHs and growth temperatures on biochemical parameters of two Cr(VI) reducing bacterial strains Pseudomonas aeruginosa Rb-1 and Ochrobactrum intermedium Rb-2 was investigated. Transmission electrone microscopy (TEM) was performed to study the intracellular distribution of Cr(VI). It was observed that initial stress of 1000 μgmL(-1) caused significant enhancement of all studied biochemical parameters at pH 7.0 and growth temperature of 37 °C showing great bioremediation potential of the strains. Transmission electron microscopy revealed that the distribution of chromium precipitates was not uniform as they were distributed in the cytoplasm as well as found associated with the periplasm and outer membrane. Fourier transform infrared spectroscopy showed the possible involvement of carboxyl, amino, sulpohonate and hydroxyl groups present on the bacterial cell surface for the binding of Cr(VI) ions. Cr(VI) stress brought about changes in the distridution of these functional groups. It can be concluded that the investigated bacterial strains adjust well to Cr(VI) stress in terms of biochemical parameters and along that exhibited alteration in morphology.

The exploitation of soil ecosystem services by agricultural management strategies requires knowledge of microbial communities in different management regimes. Crop cover by no-till management protects the soil surface, reducing the risk of erosion and nutrient leaching, but might increase straw residue-borne and soilborne plant-pathogenic fungi. A cross-site study of soil microbial communities and Fusarium fungistasis was conducted on six long-term agricultural fields with no-till and moldboard-plowed treatments. Microbial communities were studied at the topsoil surface (0 to 5 cm) and bottom (10 to 20 cm) by general bacterial and actinobacterial terminal restriction fragment length polymorphism (T-RFLP) and phospholipid fatty acid (PLFA) analyses. Fusarium culmorum soil fungistasis describing soil receptivity to plant-pathogenic fungi was explored by using the surface layer method. Soil depth had a significant impact on general bacterial as well as actinobacterial communities and PLFA profiles in no-till treatment, with a clear spatial distinction of communities (P < 0.05), whereas the depth-related separation of microbial communities was not observed in plowed fields. The fungal biomass was higher in no-till surface soil than in plowed soil (P < 0.07). Soil total microbial biomass and fungal biomass correlated with fungistasis (P < 0.02 for the sum of PLFAs; P < 0.001 for PLFA 18:2ω6). Our cross-site study demonstrated that agricultural management strategies can have a major impact on soil microbial community structures, indicating that it is possible to influence the soil processes with management decisions. The interactions between plant-pathogenic fungi and soil microbial communities are multifaceted, and a high level of fungistasis could be linked to the high microbial biomass in soil but not to the specific management strategy.

We addressed how restoration of forestry-drained peatlands affects CH(4)-cycling microbes. Despite similar community compositions, the abundance of methanogens and methanotrophs was lower in restored than in natural sites and correlated with CH(4) emission. Poor establishment of methanogens may thus explain low CH(4) emissions on restored peatlands even 10 to 12 years after restoration.

Sphingobium sp. HV3 described as an herbicide degrader harbours the pSKY4 plasmid, encoding an aromatic meta-pathway. The function of the plasmid was studied by Tn5 transposon mutagenesis and plasmid isolation and the degradation capacities of the HV3 strain were re-evaluated. Transcription of the tfdC from ortho-pathway was contrasted to the xylE and bphC of meta-pathway using real-time PCR. Cloning of the Tn5-insertion sites from the megaplasmid revealed genes for both aromatic and polyaromatic degradation. In the mutant Km24 strain the transposon was inserted to an ORF similar to the large subunit of ring hydroxylating dioxygenase, in the Km383 to a cis-biphenyl dihydrodiol dehydrogenase and in the Km187 and Km42 to a reductase component of a dioxygenase. A chlorocathecol ortho-pathway (10 kb) was amplified from the HV3 strain. The transcription of the tfdC was induced by 2,4-dichlorophenoxyacetic acid herbicide and m-xylene caused highest induction of both upper and lower aromatic meta-pathway genes. The detected novel degradation capacities (m-xylene, toluene, biphenyl, fluorene and phenanthrene) can be explained by the presence of functional meta-pathway genes in the pSKY4 megaplasmid. The characterization of the Sphingobium sp. HV3 improves our understanding of versatile catabolic bacteria unveiling roles of degradation pathways and plasmids in biodegradation.

Peatlands are a major natural source of atmospheric methane (CH4). Emissions from Sphagnum-dominated mires are lower than those measured from other mire types. This observation may partly be due to methanotrophic (i.e., methane-consuming) bacteria associated with Sphagnum. Twenty-three of the 41 Sphagnum species in Finland can be found in the peatland at Lakkasuo. To better understand the Sphagnum-methanotroph system, we tested the following hypotheses: (1) all these Sphagnum species support methanotrophic bacteria; (2) water level is the key environmental determinant for differences in methanotrophy across habitats; (3) under dry conditions, Sphagnum species will not host methanotrophic bacteria; and (4) methanotrophs can move from one Sphagnum shoot to another in an aquatic environment. To address hypotheses 1 and 2, we measured the water table and CH4 oxidation for all Sphagnum species at Lakkasuo in 1-5 replicates for each species. Using this systematic approach, we included Sphagnum spp. with narrow and broad ecological tolerances. To estimate the potential contribution of CH4 to moss carbon, we measured the uptake of delta13C supplied as CH4 or as carbon dioxide dissolved in water. To test hypotheses 2-4, we transplanted inactive moss patches to active sites and measured their methanotroph communities before and after transplantation. All 23 Sphagnum species showed methanotrophic activity, confirming hypothesis 1. We found that water level was the key environmental factor regulating methanotrophy in Sphagnum (hypothesis 2). Mosses that previously exhibited no CH4 oxidation became active when transplanted to an environment in which the microbes in the control mosses were actively oxidizing CH4 (hypothesis 4). Newly active transplants possessed a Methylocystis signature also found in the control Sphagnum spp. Inactive transplants also supported a Methylocystis signature in common with active transplants and control mosses, which rejects hypothesis 3. Our results imply a loose symbiosis between Sphagnum spp. and methanotrophic bacteria that accounts for potentially 10-30% of Sphagnum carbon.

To reveal the degradation capacity of bacteria in PAH polluted soil and rhizosphere we combined bacterial extradiol ring-cleavage dioxygenase and 16S rRNA analysis in Betula pubescens rhizoremediation. Characterisation of the functional bacterial community by RFLP revealed novel environmental dioxygenases, and their putative hosts were studied by 16S rRNA amplification. Plant rhizosphere and PAH amendment effects were detected by the RFLP/T-RFLP analysis. Functional species richness increased in the birch rhizosphere and PAH amendment impacted the compositional diversity of the dioxygenases and the structural 16S rRNA community. A shift from an Acidobacteria and Verrucomicrobia dominated to an Alpha- and Betaproteobacteria dominated community structure was detected in polluted soil. Clone sequence analysis indicated catabolic significance of Burkholderia in PAH polluted soil. These results advance our understanding of rhizoremediation and unveil the extent of uncharacterized functional bacteria to benefit bioremediation by facilitating the development of the molecular tool box to monitor bacterial populations in biodegradation., (Copyright 2009 Elsevier Ltd. All rights reserved.)

We report a new reverse primer (A621r) for use with A189f in PCR amplification of pmoA alleles in type II methanotrophs. The new primer combination was used to successfully amplify pmoA in peat monolith samples of various depths taken from fen-type peatlands in Finland. In quantitative PCR, pmoA amplicons produced from two sets of three replicate monoliths showed a significant Pearson correlation coefficient (r=0.77 and 0.61) with methane oxidation potential. The maximum methane oxidation potential and number of pmoA amplicons ranged between 8.8-40.5 micromol g (dry weight)(-1) d(-1) and 5.5 x 10(7)-18.7 x 10(7) g (wet weight)(-1), respectively, occurring in depths between 10 and 30 cm beneath the surface in the seven individual monoliths used in this study.

Northern peatlands play a major role in the global carbon cycle as sinks for CO(2) and as sources of CH(4). These diverse ecosystems develop through accumulation of partially decomposed plant material as peat. With increasing depth, peat becomes more and more recalcitrant due to its longer exposure to decomposing processes. Compared with surface peat, deeper peat sediments remain microbiologically poorly described. We detected active archaeal communities even in the deep bottom layers (-220/-280 cm) of two Finnish fen-type peatlands by 16S rRNA-based terminal restriction fragment length polymorphism analysis. In the sediments of the northern study site, all detected archaea were methanogens with Rice Cluster II (RC-II) and Methanosaetaceae as major groups. In southern peatland, Crenarchaeota of a rare unidentified cluster were present together with mainly RC-II methanogens. RNA profiles showed a larger archaeal diversity than DNA-based community profiles, suggesting that small but active populations were better visualized with rRNA. In addition, potential methane production measurements indicated methanogenic activity throughout the vertical peat profiles.

Two birch clones originating from metal-contaminated sites were exposed for 3 months to soils (sand-peat ratio 1:1 or 4:1) spiked with a mixture of polyaromatic hydrocarbons (PAHs; anthracene, fluoranthene, phenanthrene, pyrene). PAH degradation differed between the two birch clones and also by the soil type. The statistically most significant elimination (p < or = 0.01), i.e. 88% of total PAHs, was observed in the more sandy soil planted with birch, the clearest positive effect being found with Betula pubescens clone on phenanthrene. PAHs and soil composition had rather small effects on birch protein complement. Three proteins with clonal differences were identified: ferritin-like protein, auxin-induced protein and peroxidase. Differences in planted and non-planted soils were detected in bacterial communities by 16S rRNA T-RFLP, and the overall bacterial community structures were diverse. Even though both represent complex systems, trees and rhizoidal microbes in combination can provide interesting possibilities for bioremediation of PAH-polluted soils.

Methane (CH(4)) emissions from boreal wetlands show considerable seasonal variation, including small winter emissions. We addressed the seasonality of CH(4)-producing microbes by comparing archaeal communities and the rates and temperature response of CH(4) production in a boreal fen at three key phases of growing season and in winter. Archaeal community analysis by terminal restriction fragment length polymorphism and cloning of 16S ribosomal DNA and reverse-transcribed RNA revealed slight community shifts with season. The main archaeal groups remained the same throughout the year and were Methanosarcinaceae, Rice cluster II and Methanomicrobiales-associated Fen cluster. These methanogens and the crenarchaeal groups 1.1c and 1.3 were detected from DNA and RNA, but the family Methanosaetaceae was detected only from RNA. Differences between DNA- and RNA-based results suggested higher stability of DNA-derived communities and better representation of the active CH(4) producers in RNA. Methane production potential, measured as formation of CH(4) in anoxic laboratory incubations, showed prominent seasonality. The potential was strikingly highest in winter, possibly due to accumulation of methanogenic substrates, and maximal CH(4) production was observed at ca. 30 degrees C. Archaeal community size, determined by quantitative PCR, remained similar from winter to summer. Low production potential in late summer after a water level draw-down suggested diminished activity due to oxygen exposure. Our results indicated that archaeal community composition and size in the boreal fen varied only slightly despite the large fluctuations of methanogenic potential. Detection of mRNA of the methanogenic mcrA gene confirmed activity of methanogens in winter, accounting for previously reported winter CH(4) emissions.

Genes encoding key enzymes of catabolic pathways can be targeted by DNA fingerprinting to explore genetic degradation potential in pristine and polluted soils. We performed a greenhouse microcosm experiment to elucidate structural and functional bacterial diversity in polyaromatic hydrocarbon (PAH)-polluted soil and to test the suitability of birch (Betula pendula) for remediation. Degradation of PAHs was analysed by high-performance liquid chromatography, DNA isolated from soil amplified and fingerprinted by restriction fragment length polymorphism (RFLP) and terminal restriction fragment length polymorphism (T-RFLP). Bacterial 16S rRNA T-RFLP fingerprinting revealed a high structural bacterial diversity in soil where PAH amendment altered the general community structure as well as the rhizosphere community. Birch augmented extradiol dioxygenase diversity in rhizosphere showing a rhizosphere effect, and further pyrene was more efficiently degraded in planted pots. Degraders of aromatic compounds upon PAH amendment were shown by the changed extradiol ring-cleavage community structure in soil. The RFLP analysis grouped extradiol dioxygenase marker genes into 17 distinct operational taxonomic units displaying novel phylogenetic clusters of ring-cleavage dioxygenases representing putative catabolic pathways, and the peptide sequences contained conserved amino-acid signatures of extradiol dioxygenases. A branch of major environmental TS cluster was identified as being related to Parvibaculum lavantivorans ring-cleavage dioxygenase. The described structural and functional diversity demonstrated a complex interplay of bacteria in PAH pollution. The findings improve our understanding of rhizoremediation and unveil the extent of uncharacterized enzymes and may benefit bioremediation research by facilitating the development of molecular tools to detect and monitor populations involved in degradative processes.

Methanogens (domain Archaea) have a unique role in the carbon cycle as producers of the greenhouse gas methane (CH(4)). Methyl-coenzyme M reductase (MCR) is a vital enzyme in CH(4) production, and the mcrA gene coding for a subunit of MCR has been employed as a specific marker for the detection and differentiation of methanogen communities. A critical step in assessing environmental mcrA diversity is the selection of PCR primers. The objective of this study was to compare the diversity coverage of three published mcrA primer sets MCR, ME and ML (also known as MCR and Luton-mcrA) and their ability to discern methanogen communities in a drained peatland. The primers were applied to DNA extracts from unfertilised and ash-fertilised peat from two different depths. Amplified mcrA communities were cloned and sequenced, and the sequences were divided into operational taxonomic units (OTUs) by restriction fragment length polymorphism (RFLP) and sequence analysis. All primers recovered characteristic OTUs associated with the peat depths and treatments and confirmed a previous observation of low methanogen diversity. The minor differences in OTU ranges of the primers did not greatly affect the observed community composition. However, as the proportions of several OTUs varied strongly, the primers provided different quantitative representations of mcrA communities. We concluded that the ML and MCR primers had better amplification ranges than the ME set, but the use of MCR with peat samples was problematic due to poor amplification. Consequently, the ML primers were best suited for mcrA analysis of peatland methanogen communities.

For the determination of the catabolic community diversity that is related to biodegradation potential, we developed a protocol for the assessment of catabolic marker genes in polluted soils. Primers specific to upper pathway extradiol dioxygenase genes were designed which amplified a 469-bp product from Sphingomonas sp. HV3. The constructed primers were used in PCR amplification of upper pathway ring cleavage genes from DNA directly isolated from a mineral oil polluted landfill site, a mineral oil landfarming site and a birch rhizosphere-associated soil that was either artificially polluted with a PAH mixture or not polluted. Amplicons were cloned and subjected to restriction fragment length polymorphism analysis dividing the HhaI-digested products into operational taxonomic units. Altogether 26 different operational taxonomic units were detected with the sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Phylogenetic analysis divided the operational taxonomic units from the polluted soils into seven clusters. Two contained exclusively sequences with no close homologues in the database, therefore representing novel catabolic genes. This large proportion of novel extradiol sequences shows that there is an extensive unknown catabolic diversity in polluted environments.

Peat accumulating mires are important sources of the greenhouse gas methane. Methane emissions and methanogenic Archaea communities have been shown to differ between fens and bogs, implying that mire succession includes an ecological succession in methanogen communities. We investigated methane production and the methanogen communities along a chronosequence of mires (ca. 100-2,500 years), which consisted of five sites (1-5) located on the land-uplift coast of the Gulf of Bothnia. Methane production was measured in a laboratory incubation experiment. Methanogen communities were determined by amplification of a methyl coenzyme M-reductase (mcr) gene marker and analyzed by terminal-restriction fragment length polymorphism. The terminal-restriction fragment length polymorphism fingerprinting resulted in 15 terminal restriction fragments. The ordination configuration of the terminal restriction fragments data, using nonmetric multidimensional scaling, showed a clear gradient in the methanogen community structure along the mire chronosequence. In addition, fingerprint patterns of samples from the water table level and 40 cm below differed from one another in the bog site (site 5). Methane production was negligible in the three youngest fen sites (sites 1-3) and showed the highest rates in the oligotrophic fen site (site 4). Successful PCR amplification using mcr gene primers revealed the presence of a methanogen community in all five sites along the study transect.

Mires forming an ecohydrological gradient from nutrient-rich, groundwater-fed mesotrophic and oligotrophic fens to a nutrient-poor ombrotrophic bog were studied by comparing potential methane (CH(4)) production and methanogenic microbial communities. Methane production was measured from different depths of anoxic peat and methanogen communities were detected by detailed restriction fragment length polymorphism (RFLP) analysis of clone libraries, sequencing and phylogenetic analysis. Potential CH(4) production changed along the ecohydrological gradient with the fens displaying much higher production than the ombrotrophic bog. Methanogen diversity also decreased along the gradient. The two fens had very similar diversity of methanogenic methyl-coenzyme M reductase gene (mcrA), but in the upper layer of the bog the methanogen diversity was strikingly lower, and only one type of mcrA sequence was retrieved. It was related to the Fen cluster, a group of novel methanogenic sequences found earlier in Finnish mires. Bacterial 16S rDNA sequences from the fens fell into at least nine phyla, but only four phyla were retrieved from the bog. The most common bacterial groups were Deltaproteobacteria, Verrucomicrobia and Acidobacteria.

Wetlands, including peatlands, are the main source of natural methane emission. Well-defined fen microsites have different methane emissions rates, but it is not known whether the methane-producing Archaea communities vary at these sites. Possible horizontal variations of communities, in a natural oligotrophic fen, were analysed by characterizing the methanogens from two well-defined microsites: Eriophorum lawn and Hummock. Community structures were studied at two different layers of the fen, showing, respectively, high and low methane production. The structure of methanogen populations was determined using molecular techniques targeting the 16SrRNA gene and combined denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP) analysis. Results subjected to non-metric multidimensional scaling (MDS), diversity indices calculation and phylogenetic analysis revealed that upper layer communities changed with site while deeper layer communities remained the same. Phylogenetic analyses revealed six different clusters of sequences grouping with only two known orders of methanogens. Upper layers of Hummock were dominated by sequences clustering with members of Methanomicrobiales and sequences dominating the upper part of the Eriophorum lawn were related to members of the order Methanosarcinales. Novel methanogenic sequences were found at both sites at both depths. Vegetation characterizing the microsites probably influences the microbial communities in the layers of the fen where methane is produced.

The annual rate of CH4 release and potential CH4 production has recently been studied in the Salmisuo fen in eastern Finland but the microbiota responsible for the CH4 production has not been examined. The diversity of the methane producing Archaea was analysed, at different depths, in the most representative microsite (Eriophorum lawn) of the fen. Methanogen populations were studied using primers amplifying a region of the methyl-coenzyme M reductase gene. PCR products were analysed by denaturing gradient gel electrophoresis and restriction fragment length polymorphism (RFLP) analysis of clone libraries. A representative of each RFLP group was sequenced. The study revealed a change of the methanogen populations with depth. Sequences from the upper layers of the fen grouped in a novel 'Fen cluster' and were related to Methanomicrobiales. Sequences retrieved from the deeper layers of the fen were related to Methanosarcinales via the Rice Cluster-I.

The use of wood ash in forestry has been questioned because the cadmium (Cd) concentration of ash, which varies between 1 and 20 mg kg(-1) ash, exceeds the level allowed for fertilizers (3 mg kg(-1)) used in agriculture. To investigate the combined and separated effects of Cd and ash on the forest humus microflora, pumice or wood ash, spiked with a water-soluble (CdCl(2)) or -insoluble (CdO) form of Cd at three levels (0, 400 and 1000 mg kg(-1)), were applied at a fertilization level of 5000 kg ha(-1) in a laboratory microcosm study. The trial consisted of 60 microcosms (five replications per treatment), which were incubated in darkness at +20 degrees C and a constant relative air humidity of 60%. After two months the humus in the microcosms was sampled. Analyses of CO(2) evolution to measure the overall microbial activity and of phospholipid fatty acid (PLFA) pattern to measure microbial community structure were performed. The substrate-use patterns of Biolog EcoPlates were analyzed as a measure of bacterial functionality. Finally the bacterial (3)H-thymidine incorporation in the presence of different concentrations of Cd and the number of colony forming units (cfu) of bacteria on nutrient agar in the presence of 0, 5 and 20 mg Cd l(-1) agar were applied to measure Cd tolerance. The use of pumice (pH of humus under the pumice 4.0) did not induce any changes in the above variables compared to two untreated microcosms (humus pH 3.9). Pumice was therefore used to distribute the Cd evenly over the humus surface in order to estimate the possible effect of Cd without ash (pH of humus under the ash 7.0). The application of ash increased the microbial activity, changed the PLFA and substrate-use patterns and increased cfu compared to the humus under pumice. The form and level of Cd in the ash had no further effect on this result. In the humus under pumice the level, but not the form of Cd decreased the microbial activity and changed the PLFA pattern compared to the unspiked pumice. None of the treatments induced bacterial tolerance to Cd. Ash thus protected the humus microflora from the harmful effects of Cd.

Phylogenetic analyses of 16S rRNA gene sequences showed that the Gram-negative aromatic- and chloroaromatic-degrading Pseudomonas sp. strain HV3 carrying the mega-plasmid pSKY4 belongs to the genus Sphingomonas. The 16SrRNA sequence is most related to Sphingomonas chlorophenolica strains ATCC 33790(T) (98.5%) and SR3 (98.4%) and Sphingomonas sp. SS86 (98.4%). The G+C content was 64 mol%, and the DNA-DNA hybridization-based relative homology of strain HV3 to the S. chlorophenolica ATCC 33790(T) and S. chlorophenolica RA2 was 59.6% and 35.9%, respectively. The results showed that although strain HV3 is related to S. chlorophenolica it differs in certain characteristics. It is therefore proposed to reclassify Pseudomonas sp. strain HV3 as Sphingomonas sp. HV3.

Pseudomonas sp. strain HV3 degrades aromatics and chloroaromatics. It harbours a mega-plasmid, designated pSKY4, from which the gene cmpE, encoding a catechol 2,3-dioxygenase (C23O) catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde, was cloned and sequenced. The deduced amino acid (aa) sequence shows the highest homology, 52%, to the deduced aa sequences of xylE1 and dmpB. The deduced 307-aa sequence of cmpE contains the extradiol ring-cleavage signature in the same position as other 307-aa C23O-encoding genes.