Cumulative effects of natural and anthropogenic disturbances on the forest carbon balance in the oil sands region of Alberta, Canada; a pilot study (1985-2012).

BACKGROUND: Assessing cumulative effects of anthropogenic and natural disturbances on forest carbon (C) stocks and fluxes, because of their relevance to climate change, is a requirement of environmental impact assessments (EIAs) in Canada. However, tools have not been developed specifically for these purposes, and in particular for the boreal forest of Canada, so current forest C assessments in EIAs take relatively simple approaches. Here, we demonstrate how an existing tool, the Generic Carbon Budget Model (GCBM), developed for national and international forest C reporting, was used for an assessment of the cumulative effects of anthropogenic and natural disturbances to support EIA requirements. We applied the GCBM to approximately 1.3 million ha of upland forest in a pilot study area of the oil sands region of Alberta that has experienced a large number of anthropogenic (forestry, energy sector) and natural (wildfire, insect) disturbances. RESULTS: Over the 28 years, 25% of the pilot study area was disturbed. Increasing disturbance emissions, combined with declining net primary productivity and reductions in forest area, changed the study area from a net C sink to a net C source. Forest C stocks changed from 332.2 Mt to 327.5 Mt, declining by 4.7 Mt at an average rate of 0.128 tC ha-1 yr-1. The largest cumulative areas of disturbance were caused by wildfire (139,000 ha), followed by the energy sector (110,000 ha), insects (33,000 ha) and harvesting (31,000 ha) but the largest cumulative disturbance emissions were caused by the energy sector (9.5 Mt C), followed by wildfire (5.5 Mt C), and then harvesting (1.3 Mt C). CONCLUSION: An existing forest C model was used successfully to provide a rigorous regional cumulative assessment of anthropogenic and natural disturbances on forest C, which meets requirements of EIAs in Canada. The assessment showed the relative importance of disturbances on C emissions in the pilot study area, but their relative importance is expected to change in other parts of the oil sands region because of its diversity in disturbance types, patterns and intensity. Future assessments should include peatland C stocks and fluxes, which could be addressed by using the Canadian Model for Peatlands.

Spatially-integrated estimates of net ecosystem exchange and methane fluxes from Canadian peatlands.

BACKGROUND: Peatlands are an important component of Canada's landscape, however there is little information on their national-scale net emissions of carbon dioxide [Net Ecosystem Exchange (NEE)] and methane (CH4). This study compiled results for peatland NEE and CH4 emissions from chamber and eddy covariance studies across Canada. The data were summarized by bog, poor fen and rich-intermediate fen categories for the seven major peatland containing terrestrial ecozones (Atlantic Maritime, Mixedwood Plains, Boreal Shield, Boreal Plains, Hudson Plains, Taiga Shield, Taiga Plains) that comprise > 96% of all peatlands nationally. Reports of multiple years of data from a single site were averaged and different microforms (e.g., hummock or hollow) within these peatland types were kept separate. A new peatlands map was created from forest composition and structure information that distinguishes bog from rich and poor fen. National Forest Inventory k-NN forest structure maps, bioclimatic variables (mean diurnal range and seasonality of temperatures) and ground surface slope were used to construct the new map. The Earth Observation for Sustainable Development map of wetlands was used to identify open peatlands with minor tree cover. RESULTS: The new map was combined with averages of observed NEE and CH4 emissions to estimate a growing season integrated NEE (± SE) at - 108.8 (± 41.3) Mt CO2 season-1 and CH4 emission at 4.1 (± 1.5) Mt CH4 season-1 for the seven ecozones. Converting CH4 to CO2 equivalent (CO2e; Global Warming Potential of 25 over 100 years) resulted in a total net sink of - 7.0 (± 77.6) Mt CO2e season-1 for Canada. Boreal Plains peatlands contributed most to the NEE sink due to high CO2 uptake rates and large peatland areas, while Boreal Shield peatlands contributed most to CH4 emissions due to moderate emission rates and large peatland areas. Assuming a winter CO2 emission of 0.9 g CO2 m-2 day-1 creates an annual CO2 source (24.2 Mt CO2 year-1) and assuming a winter CH4 emission of 7 mg CH4 m-2 day-1 inflates the total net source to 151.8 Mt CO2e year-1. CONCLUSIONS: This analysis improves upon previous basic, aspatial estimates and discusses the potential sources of the high uncertainty in spatially integrated fluxes, indicating a need for continued monitoring and refined maps of peatland distribution for national carbon and greenhouse gas flux estimation.

Estimation of snag carbon transfer rates by ecozone and lead species for forests in Canada.

Standing dead trees (snags) and downed woody debris contribute substantially to the carbon (C) budget of Canada's forest. Accurate parameterization of the C transfer rates (CTRs) from snags to downed woody debris is important for forest C dynamics models such as the Carbon Budget Model of the Canadian Forest Sector (CBM-CFS3), but CTRs are rarely measured or reported in the literature. Therefore, forest C models generally use snag fall rates (FRs) available in the literature, as a proxy for CTRs. However, FRs are based on stem counts while CTRs refer to mass transfers. Stem mass and stem number are not linearly related, with small diameter trees representing disproportionately lower C mass transfers. Therefore this proxy, while convenient, may bias C transfer from standing dead to downed woody material. Here, we combined tree data from 10802 sample plots and previously published species-specific individual-tree relationships between tree diameter (diameter at breast height, dbh) and fall rate to derive stand-level estimates of CTRs for the CBM-CFS3. We estimated CTRs and FRs and used the FR values to validate this approach by comparing them with standardized FR values compiled from the literature. FRs generally differed from CTRs. The overall CTR (4.78% +/- 0.02% per year, mean +/- SE) was significantly smaller than the overall FR (5.40% +/- 0.02% per year; mean +/- SE). Both the difference between FR and CTR (FR - CTR) and the CTR itself varied by ecozone, with ecozone means for CTR ranging from 3.94% per year to 10.02% per year. This variation was explained, in part, by heterogeneity in species composition, size (dbh distribution), structure, and age of the stands. The overall mean CTR estimated for the Snag_Stemwood (4.78% per year) and the Snag_Branches (11.95% per year) pools of the CBM-CFS3 were approximately 50% and 20% higher than the current default rates used in the CBM-CFS3 of 3.2% and 10.0%, respectively. Our results demonstrate that using CTRs to estimate the annual C transfer from standing dead trees to downed woody biomass will yield more accurate estimates of C fluxes than using a FR proxy, and this accuracy could be further improved by accounting for differences in ecozone, stand component (hardwood or softwood), or lead species.

Microbial degradation of the biocide polyhexamethylene biguanide: isolation and characterization of enrichment consortia and determination of degradation by measurement of stable isotope incorporation into DNA.

AIMS: To isolate micro-organisms capable of utilizing polyhexamethylene biguanide (PHMB) as a sole source of nitrogen, and to demonstrate biodegradation of the biocide. METHODS AND RESULTS: Two consortia of bacteria were successfully enriched at the expense of PHMB, using sand from PHMB-treated swimming pools as inoculum. Both consortia were shown to contain bacteria belonging to the genera Sphingomonas, Azospirillum and Mesorhizobium. It was shown that the presence of both Sphingomonas and Azospirillum spp. was required for extensive growth of the consortia. In addition, the Sphingomonads were the only isolates capable of growth in axenic cultures dosed with PHMB. Using a stable isotope (15N)-labelled PHMB, metabolism of the biocide by both consortia was demonstrated. By comparing the level of 15N atom incorporation into bacterial DNA after growth on either 15N-PHMB or 15N-labelled NH4Cl, it was possible to estimate the percentage of PHMB biodegradation. CONCLUSIONS: The microbial metabolism of nitrogen from the biguanide moiety of PHMB has been demonstrated. It was revealed that Sphingomonas and Azospirillum spp. are the principal organisms responsible for growth at the expense of PHMB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the microbial metabolism of PHMB.

A chemotaxis cluster from Agrobacterium tumefaciens.

We report the DNA sequence of a 9.6-kb region of the Agrobacterium tumefaciens chromosome containing a putative 8-kb chemotaxis operon. The putative operon begins with orf1, whose predicted protein product shows strong sequence identity to methyl-accepting chemotaxis proteins (MCPs), followed by orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, orf10. All of the identified homologues show a high degree of sequence conservation with their counterparts in the che operons from Sinorhizobium meliloti and Rhodobacter sphaeroides, and are arranged in a similar order. Mutations in orf1 and cheA result in impaired chemotaxis, whereas deletion of orf10, appears to have no effect on chemotaxis or motility. Although the putative operon does not contain a cheW homologue, heterologous probing and PCR using consensus primers indicates that cheW maps elsewhere in the Agrobacterium genome.

Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure.

We report the DNA sequence of 7205 bp of the Agrobacterium tumefaciens chromosome. This contains a putative operon encoding homologues of the flagellar rod and associated proteins FlgBCG and FliE, the L and P ring proteins (FlgHI) a possible flagellum-specific export protein FliP, and two proteins of unknown function, FlgA and FliL. Several of these genes have overlapping stop and start codons. Three non-flagellate Tn5-induced mutations map to this operon: fla-11 to the first gene, encoding the rod protein FlgB; fla-15 to flgA; and fla-12 to fliL. A site-specific mutation introduced into the final gene in this cluster, fliP, also resulted in a non-flagellate phenotype. This indicates that the operon is expressed, and that at least FlgB, FlgA, FliL and FliP are required for flagellar assembly in A. tumefaciens. The bulk of this operon is conserved in the same order in Rhizobium meliloti.

The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN.

We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly.

Timely culture for mycobacteria which utilizes a microcolony method.

For the isolation of mycobacteria from clinical specimens, we evaluated a method that used a thinly poured Middlebrook 7H11 agar plate (10 by 90 mm) that was examined microscopically. Inoculated plates were sealed, incubated, and examined at regular intervals for the appearance of microcolonies. Plates were examined microscopically, while still sealed, by focusing on the agar surface through the bottom of the plate and the agar. Plates were scanned at low power (x40 total magnification), and colony morphology was confirmed at intermediate power (x100 to x180 magnification). This method was compared with a traditional method that used macroscopic examination of standard mycobacterial media. By using all specimens submitted for mycobacterial culture over the duration of the study, the method was evaluated until 270 isolates of mycobacteria (Mycobacterium tuberculosis, n = 103; M. avium-M. intracellulare, n = 115; miscellaneous, n = 52) were detected. While the conventional method required an average of 23 days to the time of first detection of mycobacteria, the experimental method required an average of only 11 days. When limited to acid-fast stain-positive specimens that were culture positive for M. tuberculosis, the average interval to positivity was 7 days for the microcolony method compared with 17 days for the conventional method. With the experimental method, the microscopic colonial morphology allowed for the presumptive identification of M. tuberculosis colonies, which were distinguished by cording, and M. avium-M. intracellulare colonies, which were smooth and entire. Presumptive identification was complete for 83.5% of the M. tuberculosis isolates within 10 days and for 85% of the M. avium-M. intracellulare isolates within 11 days after inoculation. If the microcolony method was combined with a conventional tube medium, the composite would optimize for speed of recovery while providing the full sensitivity of the conventional method. In addition to reducing the interval to positivity, the microcolony method allows for the easy detection of mixed mycobacterial infections and yields a presumptive identification that facilitates the selection of a confirmatory gene probe test.

Sequence of an oleosin cDNA from Brassica napus.

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived lambda gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 bp and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3' untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.

Cloning and characterisation of an oleosin gene from Brassica napus.

The sequence of an oleosin gene from Brassica napus has been determined. This gene contains a single intron of 437 bp and encodes a polypeptide of 195 amino acids. The oleosin gene product has an estimated molecular mass of 21.5 kDa and consists of a highly hydrophobic central domain flanked by relatively polar N- and C-terminal domains. The central domain is highly conserved between all oleosins sequenced to date and contains a run of periodically spaced leucine residues similar to that of a leucine-zipper motif. The gene has been shown to be expressed specifically in the embryo, maximally between 9 and 11 weeks after flowering, i.e. during the seed desiccation stage. Two transcriptional start sites have been mapped to -70 and -21 of the ATG and a putative ABA-responsive element and three repeated motifs have been identified in the promoter. These short promoter sequences could correspond to regulatory elements responsible for embryo-specific gene expression. Up to six genes exist in the oleosin gene family.

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins.

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.

Swimming against the tide: chemotaxis in Agrobacterium.

Chemotaxis in bacteria is an excellent model for signal transduction processes. In Agrobacterium tumefaciens, the causative agent of crown gall tumour on wounded plants, it is a vital part of the organism's biology. A chromosomally-determined chemotaxis system causes the bacterium to be attracted into the rhizosphere by chemoattractants in plant exudates. By interfacing with this system, the multifunctional products of two Ti-plasmid encoded genes, virA and virG, allow the sensing of specific wound phenolics such as acetosyringone. This attracts Ti-plasmid harbouring A. tumefaciens to wound sites, where the higher acetosyringone concentrations lead to virA and virG-mediated induction of the vir-genes. The products of the induced genes, act in concert to effect transfer of the T-DNA to the plant cell.

Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.

Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens.

virA and virG are the Ti-plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone.

Octopine and nopaline Ti-plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir-inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti-plasmids with Tn5 insertions in virB, C, D or E exhibited marked chemotaxis towards acetosyringone. However, Ti-plasmids with mutations in virA or virG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir-induction.

Light regulation of a SSRubisco-nos chimaeric gene: photoregulatory control sequences from a C3 plant function in cells of a CAM plant.

A SSRubisco-nos chimaeric gene has been constructed, in an oncogenic Ti-plasmid vector. A 900bp soybean SSRubisco upstream fragment, carrying CAAT and TATA boxes and transcription initiation point, was fused to the nos coding region, the fusion site being within the 5'-untranslated region. When this chimaeric construct was transferred to Kalanchoe cells, nos expression was shown to be light-regulated. Thus DNA sequences responsible for light-dark control of gene expression are wholly or partly contained within the 900bp soybean SSRubisco upstream region. Moreover, this is the first demonstration that photoregulatory elements in a gene derived from a C3 plant, function in cells of a plant exhibiting the CAM trait.

A functional map of the nopaline synthase promoter.

This paper describes the first functional map of a promoter expressed from the plant chromosome. We have constructed a series of overlapping deletion mutants within the region upstream of the Ti-plasmid encoded nopaline synthase (nos) gene. By monitoring nos expression in tumour tissue we have inferred a functional map of the nos promoter. The maximum length of sequence upstream of the transcription initiation point required to express wild type levels of nopaline synthase is 88 bp. Within this region, the "CAAT" box is essential for maximal activity; deletion of this sequence reduced apparent nos expression by over 80%. Presence of an intact or partial "TATA" box in the absence of the "CAAT" box supports a barely detectable level of nopaline synthase. Removal of all sequences upstream of the nos coding sequence results in no detectable activity.