Decrypting Molecular Mechanisms Involved in Counteracting Copper and Nickel Toxicity in Jack Pine (Pinus banksiana) Based on Transcriptomic Analysis.

The remediation of copper and nickel-afflicted sites is challenged by the different physiological effects imposed by each metal on a given plant system. Pinus banksiana is resilient against copper and nickel, providing an opportunity to build a valuable resource to investigate the responding gene expression toward each metal. The objectives of this study were to (1) extend the analysis of the Pinus banksiana transcriptome exposed to nickel and copper, (2) assess the differential gene expression in nickel-resistant compared to copper-resistant genotypes, and (3) identify mechanisms specific to each metal. The Illumina platform was used to sequence RNA that was extracted from seedlings treated with each of the metals. There were 449 differentially expressed genes (DEGs) between copper-resistant genotypes (RGs) and nickel-resistant genotypes (RGs) at a high stringency cut-off, indicating a distinct pattern of gene expression toward each metal. For biological processes, 19.8% of DEGs were associated with the DNA metabolic process, followed by the response to stress (13.15%) and the response to chemicals (8.59%). For metabolic function, 27.9% of DEGs were associated with nuclease activity, followed by nucleotide binding (27.64%) and kinase activity (10.16%). Overall, 21.49% of DEGs were localized to the plasma membrane, followed by the cytosol (16.26%) and chloroplast (12.43%). Annotation of the top upregulated genes in copper RG compared to nickel RG identified genes and mechanisms that were specific to copper and not to nickel. NtPDR, AtHIPP10, and YSL1 were identified as genes associated with copper resistance. Various genes related to cell wall metabolism were identified, and they included genes encoding for HCT, CslE6, MPG, and polygalacturonase. Annotation of the top downregulated genes in copper RG compared to nickel RG revealed genes and mechanisms that were specific to nickel and not copper. Various regulatory and signaling-related genes associated with the stress response were identified. They included UGT, TIFY, ACC, dirigent protein, peroxidase, and glyoxyalase I. Additional research is needed to determine the specific functions of signaling and stress response mechanisms in nickel-resistant plants.

Gene expression profiling of Jack Pine (Pinus banksiana) under copper stress: Identification of genes associated with copper resistance.

Understanding the genetic response of plants to copper stress is a necessary step to improving the utility of plants for environmental remediation and restoration. The objectives of this study were to: 1) characterize the transcriptome of Jack Pine (Pinus banksiana) under copper stress, 2) analyze the gene expression profile shifts of genotypes exposed to copper ion toxicity, and 3) identify genes associated with copper resistance. Pinus banksiana seedlings were treated with 10 mmoles of copper and screened in a growth chamber. There were 6,213 upregulated and 29,038 downregulated genes expressed in the copper resistant genotypes compared to the susceptible genotypes at a high stringency based on the false discovery rate (FDR). Overall, 25,552 transcripts were assigned gene ontology. Among the top upregulated genes, the response to stress, the biosynthetic process, and the response to chemical stimuli terms represented the highest proportion of gene expression for the biological processes. For the molecular function category, the majority of expressed genes were associated with nucleotide binding followed by transporter activity, and kinase activity. The majority of upregulated genes were located in the plasma membrane while half of the total downregulated genes were associated with the extracellular region. Two candidate genes associated with copper resistance were identified including genes encoding for heavy metal-associated isoprenylated plant proteins (AtHIP20 and AtHIP26) and a gene encoding the pleiotropic drug resistance protein 1 (NtPDR1). This study represents the first report of transcriptomic responses of a conifer species to copper ions.

Transcriptome Analysis Reveals Changes in Whole Gene Expression, Biological Process, and Molecular Functions Induced by Nickel in Jack Pine (Pinus banksiana).

Understanding the genetic response of plants to nickel stress is a necessary step to improving the utility of plants in environmental remediation and restoration. The main objective of this study was to generate whole genome expression profiles of P. banksiana exposed to nickel ion toxicity compared to reference genotypes. Pinus banksiana seedlings were screened in a growth chamber setting using a high concentration of 1600 mg of nickel per 1 kg of soil. RNA was extracted and sequenced using the Illumina platform, followed by de novo transcriptome assembly. Overall, 25,552 transcripts were assigned gene ontology. The biological processes in water-treated samples were analyzed, and 55% of transcripts were distributed among five categories: DNA metabolic process (19.3%), response to stress (13.3%), response to chemical stimuli (8.7%), signal transduction (7.7%) and response to biotic stimulus (6.0%). For molecular function, the highest percentages of genes were involved in nucleotide binding (27.6%), nuclease activity (27.3%) and kinase activity (10.3%). Sixty-two percent of genes were associated with cellular compartments. Of these genes, 21.7% were found in the plasma membrane, 16.1% in the cytosol, 12.4% with the chloroplast and 11.9% in the extracellular region. Nickel ions induced changes in gene expression, resulting in the emergence of differentially regulated categories. Overall, there were significant changes in gene expression with a total 4128 genes upregulated and 3754 downregulated genes detected in nickel-treated genotypes compared to water-treated control plants. For biological processes, the highest percentage of upregulated genes in plants exposed to nickel were associated with the response to stress (15%), the response to chemicals (11,1%), carbohydrate metabolic processes (7.4%) and catabolic processes (7.4%). The largest proportions of downregulated genes were associated with the biosynthetic process (21%), carbohydrate metabolic process (14.3%), response to biotic stimulus (10.7%) and response to stress (10.7%). For molecular function, genes encoding for enzyme regulatory and hydrolase activities represented the highest proportion (61%) of upregulated gene. The majority of downregulated genes were involved in the biosynthetic processes. Overall, 58% of upregulated genes were located in the extracellular region and the nucleus, while 42% of downregulated genes were localized to the plasma membrane and 33% to the extracellular region. This study represents the first report of a transcriptome from a conifer species treated with nickel.

Transcriptome analysis of trembling aspen (Populus tremuloides) under nickel stress.

Plants have evolved heavy metal tolerance mechanisms to adapt and cope with nickel (Ni) toxicity. Decrypting whole gene expression of Trembling Aspen (Pinus tremuloides) under nickel stress could elucidate the nickel resistance/tolerance mechanisms. The main objectives of the present research were to 1) characterize the P. tremuloides transcriptome, and 2) compare gene expression dynamics between nickel-resistant and nickel-susceptible P. tremuloides genotypes with Whole Transcriptome (WT) sequencing. Illumina Sequencing generated 27-45 million 2X150 paired-end reads of raw data per sample. The alignment performed with StringTie Software added two groups of transcripts to the draft genome annotation. One group contained 32,677 new isoforms that match to 17,254 genes. The second group contained 17,349 novel transcripts that represent 16,157 novel genes. Overall, 52,987 genes were identified from which 36,770 genes were selected as differently expressed. With the high stringency (two-fold change, FDR value ≤ 0.05 and logFC value ≥1 (upregulated) or ≤ -1 (downregulated), after GSEA analysis and filtering for gene set size, 575 gene sets were upregulated and 146 were downregulated in nickel resistant phenotypes compared to susceptible genotypes. For biological process, genes associated with translation were significantly upregulated while signal transduction and cellular protein process genes were downregulated in resistant compared to susceptible genotypes. For molecular function, there was a significant downregulation of genes associated with DNA binding in resistant compared to susceptible lines. Significant upregulation was observed in genes located in ribosome while downregulation of genes in chloroplast and mitochondrion were preponderant in resistant genotypes compared to susceptible. Hence, from a whole transcriptome level, an upregulation in ribosomal and translation activities was identified as the main response to Ni toxicity in the resistant plants. More importantly, this study revealed that a metal transport protein (Potrs038704g29436 -ATOX1-related copper transport) was among the top upregulated genes in resistant genotypes when compared to susceptible plants. Other identified upregulated genes associated with abiotic stress include genes coding for Dirigent Protein 10, GATA transcription factor, Zinc finger protein, Auxin response factor, Bidirectional sugar transporter, and thiamine thiazole synthase.

DNA methylation and histone modifications induced by abiotic stressors in plants.

BACKGROUND: A review of research shows that methylation in plants is more complex and sophisticated than in microorganisms and animals. Overall, studies on the effects of abiotic stress on epigenetic modifications in plants are still scarce and limited to few species. Epigenetic regulation of plant responses to environmental stresses has not been elucidated. This study summarizes key effects of abiotic stressors on DNA methylation and histone modifications in plants. DISCUSSION: Plant DNA methylation and histone modifications in responses to abiotic stressors varied and depended on the type and level of stress, plant tissues, age, and species. A critical analysis of the literature available revealed that 44% of the epigenetic modifications induced by abiotic stressors in plants involved DNA hypomethylation, 40% DNA hypermethylation, and 16% histone modification. The epigenetic changes in plants might be underestimated since most authors used methods such as methylation-sensitive amplification polymorphism (MSAP), High performance liquid chromatography (HPLC), and immunolabeling that are less sensitive compared to bisulfite sequencing and single-base resolution methylome analyses. More over, mechanisms underlying epigenetic changes in plants have not yet been determined since most reports showed only the level or/and distribution of DNA methylation and histone modifications. CONCLUSIONS: Various epigenetic mechanisms are involved in response to abiotic stressors, and several of them are still unknown. Integrated analysis of the changes in the genome by omic approaches should help to identify novel components underlying mechanisms involved in DNA methylation and histone modifications associated with plant response to environmental stressors.

Rhizobioaugmentation of Casuarina glauca with N-fixing actinobacteria Frankia decreases enzymatic activities in wastewater irrigated soil: effects of Frankia on C. glauca growth.

The use of wastewater for irrigation in agroforestry is cost-effective for water management. It is well established that rhizospheric microorganisms such as N2-fixing bacteria are able to modulate rhizobioaugmention and to boost phyoremediation process. To date, no study has been conducted to evaluate biological effects of rhizobioaugmentation in Casuarina glauca trees induced by their symbiont N-fixing actinobacteria of the genus Frankia. The objective of the present study was to evaluate the main effects of rhizobioaugmentation on the biological activity in the C. glauca's rhizosphere and on C. glauca growth in soils irrigated with industrial wastewater. Two Frankia strains (BMG5.22 and BMG5.23) were used in a single or dual inoculations of C. glauca seedlings irrigated with industrial wastewater. Soil enzymes activity related to carbon, phosphorus, sulfur and nitrogen cycling were measured. Results revealed that the BMG5.22 Frankia strain increases significantly the size (dry weight) of C. glauca shoots and roots while dual inoculation increased significantly the root length. Surprisingly, ß-glucosidase (BG), cellobiohydrolase (CBH), ß-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), leucine aminopeptidase (LAP), and peroxidase (PER) activity in the rhizosphere decreased significantly in soils treated with the two strains of symbionts. This suggests no positive correlations between enzymatic activity and C. glauca growth.

Metal Toxicity and Resistance in Plants and Microorganisms in Terrestrial Ecosystems.

Metals are major abiotic stressors of many organisms, but their toxicity in plants is not as studied as in microorganisms and animals. Likewise, research in plant responses to metal contamination is sketchy. Candidate genes associated with metal resistance in plants have been recently discovered and characterized. Some mechanisms of plant adaptation to metal stressors have been now decrypted. New knowledge on microbial reaction to metal contamination and the relationship between bacterial, archaeal, and fungal resistance to metals has broadened our understanding of metal homeostasis in living organisms. Recent reviews on metal toxicity and resistance mechanisms focused only on the role of transcriptomics, proteomics, metabolomics, and ionomics. This review is a critical analysis of key findings on physiological and genetic processes in plants and microorganisms in responses to soil metal contaminations.

Development and characterization of species-diagnostic ISSR and SCAR DNA markers for differentiating red maple (Acer rubrum) and silver maple (A. saccharinum).

Red maple (Acer rubrum) and silver maple (A. saccharinum) are sister species that readily hybridize in nature. No genetic or barcoding markers have been tested in these species. The main objective of the present study is to develop and characterize molecular markers for distinguishing A. rubrum and A. saccharinum and to validate the hybridity of A. freemanii derived from their crossings using the ISSR marker system. Thirteen A. rubrum and seven A. saccharinum populations were used. Four ISSR primers including ISSR 5, ISSR 8, ISSR 10, and ISSR UBC 825 were selected to amplify genomic DNA from the two species and their hybrids. Each primer generated at least one species-diagnostic ISSR marker for a total of six. Analysis of A. freemanii collected from North Dakota (USA) confirmed that the genotypes screened were true hybrids between A. rubrum and A. saccharinum. These markers were cloned and sequenced. Successful sequences were converted to SCAR markers using specifically designed primers. Overall, the developed diagnostic and specific ISSR and SCAR markers are useful in the certification of these two maple species and their hybrids. They can be used in tracking the introgression of A. rubrum and A. saccharinum DNA in other hybrid trees or populations.

Characterization of chloroplast genomes of Alnus rubra and Betula cordifolia, and their use in phylogenetic analyses in Betulaceae.

OBJECTIVE: Betulaceae is a relatively small birch family that comprises about 160 deciduous trees and shrubs. Chloroplast (cp) genome sequencing of Alnus rubra and Betula cordifolia was carried out to elucidate their molecular features and phylogenetic relationship among species in Betulaceae family. METHODS: Chloroplast genome sequencing was carried out using next generation sequencing method. Molecular and genomic features of the two cp genomes were characterized with other cp genomes in Betulaceae. Also, molecular phylogenetic analysis was performed using the whole cp genome sequences. RESULTS: The average cp genome length was 160,136 bp among the Betulaceae species. Base compositions of the cp genomes were skewed toward a high AT ratio, with an average of 63.4%. We identified 117 different genes 83 with protein coding, 4 with ribosomal RNA, and 30 with tRNA. Eighteen genes contained introns which were conserved among the cp genomes of all Betulaceae. We mined 82 SSRs from the cp genomes of A. rubra, A. cordifolia, and A. nana. The SSRs were variable in motif repeat numbers and presence/absence among the cp genomes. CONCLUSION: Chloroplast genome-wide sequence comparison from 11 Betulaceae species and one cp genome of evergreen oak revealed that the patterns of sequence variations were congruent with two subfamily classification Betuloideae (Alnus and Betula) and Corylaceae (Corylus, Ostrya, and Carpinus). Subsequent phylogenetic analysis also supports the sub-classifications of these species.

Differential effects of nickel dosages on in vitro and in vivo seed germination and expression of a high affinity nickel-transport family protein (AT2G16800) in trembling aspen (Populus tremuloides).

It has been demonstrated that a number of metals including mercury (Hg), zinc (Zn), cadmium (Cd), cobalt (Co), lead (Pb), copper (Cu), and nickel (Ni) decrease seed germination rates and plant growth. The threshold levels of metal toxicity on seed germination, plant development, and gene regulation have not been studied in detail. The main objective of this study was to assess in vitro and in vivo the effects of different doses of nickel on Trembling aspen (Populus tremuloides) seed germination and regulation of the high affinity nickel transporter family protein (AT2G16800) gene. The in vitro assays showed that Nickel completely inhibited seed germination even at the lowest concentration of 0.401 mg Ni per mL (in media) tested. However, when the same concentration of nickel (150 mg Ni per 1 kg of dry soil) was added to soil samples, during the vivo assays, almost all of the seeds germinated. Significant inhibition of seed germination was observed when soil samples were treated with at least 400 mg/kg of Ni. No damages were observed on growing seedlings treated with 150, 400, and 800 mg/kg of Ni. Only the highest dose of 1, 600 mg/kg resulted in visible leaf and stem damages and reduced growth on 75% of seedlings. A significant repression of the AT2G16800 gene was observed for the 400, 800, and 1600 mg/kg of nickel treatments compared to the water control with the lowest level of expression observed in samples treated with 800 mg/kg of Ni. Results of this study suggest that P. tremuloides populations will likely be sustainable for long term in sites that are highly contaminated with Ni including mining regions since the bioavailable amount of this metal is usually below 400 mg/kg in Canada.

Differential levels of gene expression and molecular mechanisms between red maple (Acer rubrum) genotypes resistant and susceptible to nickel toxicity revealed by transcriptome analysis.

Knowledge of regulation of genes associated with metal resistance in higher plants is very limited. Many plant species have developed different genetic mechanisms and metabolic pathways to cope with metal toxicity. The main objectives of this study were to 1) assess gene expression dynamics of A. rubrum in response to nickel (Ni) stress and 2) describe gene function based on ontology. Certified A. rubrum genotypes were treated with 1,600 mg of Ni per 1 Kg of soil corresponding to a soil total nickel content in a metal-contaminated region in Ontario, Canada. Nickel resistant and susceptible genotypes were selected and used for transcriptome analysis. Overall, 223,610,443 bases were generated. Trinity reads were assembled to trinity transcripts. The transcripts were mapped to protein sequences and after quality controls and appropriate trimmings, 66,783 annotated transcripts were selected as expressed among the libraries. The study reveals that nickel treatment at a high dose of 1,600 mg/kg triggers regulation of several genes. When nickel-resistant genotypes were compared to water controls, 6,263 genes were upregulated and 3,142 were downregulated. These values were 3,308 and 2,176, respectively, when susceptible genotypes were compared to water control. The coping mechanism of A. rubrum to Ni toxicity was elucidated. Upregulation of genes associated with transport in cytosol was prevalent in resistant genotypes compared to controls while upregulation of genes associated with translation in the ribosome was higher in susceptible genotypes when compared to water. The analysis revealed no major gene associated with Ni resistance in A. rubrum. Overall, the results of this study suggest that the genetic mechanism controlling the resistance of this species to nickel is controlled by genes with limited expression. The subtle differences between resistant and susceptible genotypes in gene regulation were detected using water-treated genotypes as references.

Retrotransposons in Betula nana, and interspecific relationships in the Betuloideae, based on inter-retrotransposon amplified polymorphism (IRAP) markers.

The Betulaceae family comprises two subfamilies, Betuloideae and Corylaceae. The subfamily Betuloideae contains two genera, Alnus Mill. and Betula L. Twenty putative long terminal repeat (LTR) retrotransposons were mined from 171 scaffolds containing 5,208,995 bp of dwarf birch (Betula nana) genome sequences. Five retrotransposons were finally selected after filtering the retrotransposon canonical features and nucleotide similarities between left and right LTR sequences. Of the five retroelements, three elements were found to be Ty1/Copia retrotransposons; identity of the other two elements could not be ascertained due to sequence undetermined 'N' bases in the sequence database. Inter-retrotranposon amplified polymorphism (IRAP) analysis, based on the LTR sequences of the mined LTR-retrotransposons, produced 179 discernible IRAP bands among the Alnus and Betula genera. Sequence analysis revealed no size homoplasy among the homologous IRAP bands. Phylogenetic and principle coordinate analysis, based on the band sharing among the taxa, showed the species in two different genera were clearly separated. The subgenera in each genus of Alnus and Betula were also distinguishable from the IRAP profiles. In the genus Betula, the species in subgenus Betula showed mixed clustering between species. This is incongruent with the phylogeographical distribution of the species.

Expression of Genes Associated with Nickel Resistance in Red Oak (Quercus rubra) Populations from a Metal Contaminated Region.

Although many studies have reported mechanisms of resistance to metals in herbaceous species, there is very little information on metal coping strategy in hardwood species such as Quercus rubra. The main objective of this study was to determine the expression of genes associated with nickel resistance in red oak (Q. rubra) populations from metal contaminated and uncontaminated sites in the Northern Ontario. Six genes associated with nickel resistances in model and non-model plants were targeted. Differential expressions of these genes were observed in Q. rubra from all the sites, but association between metal contamination and gene expression was not established. This suggests that the bioavailable amounts of metals found in metal contaminated soils in mining sites in northern Ontario and likely in many mining regions around the world cannot trigger a genetic response in higher plant species.

Changes in enzymatic activities in metal contaminated and reclaimed lands in Northern Ontario (Canada).

Metal and sulfur dioxide (SO2) contaminations in Northern Ontario (Canada), especially in the Greater Sudbury Region (GSR) caused by mining activities have resulted in severe environmental degradations. A long term restoration program has led to significant landscape changes and healthy ecosystems. The objective of this study was to assess variation in enzymatic activities and soil respiration in metal contaminated and reclaimed ecosystems. Soil analysis revealed that respiration rates were higher in metal contaminated limed soils (65ppm) compared to adjacent unlimed areas (35ppm). The respiration rates in metal contaminated sites (55ppm) were significantly lower compared to reference (metal-uncontaminated) areas (90ppm). ß-glucosidase (BG), cellobiohydrolase (CBH), ß-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), and leucine aminopeptidase (LAP) activities were significantly higher in limed compared to unlimed sites. Metal contamination significantly reduced the activities of these enzymes with the exception of LAP. An opposite trend was observed for peroxidase (PER) activity that was lower in limed compared to corresponding unlimed areas. Likewise, PER activity values were significantly lower in metal contaminated than in uncontaminated reference sites.

Microbial Response to Soil Liming of Damaged Ecosystems Revealed by Pyrosequencing and Phospholipid Fatty Acid Analyses.

AIMS: To assess the effects of dolomitic limestone applications on soil microbial communities' dynamics and bacterial and fungal biomass, relative abundance, and diversity in metal reclaimed regions. METHODS AND RESULTS: The study was conducted in reclaimed mining sites and metal uncontaminated areas. The limestone applications were performed over 35 years ago. Total microbial biomass was determined by Phospholipid fatty acids. Bacterial and fungal relative abundance and diversity were assessed using 454 pyrosequencing. There was a significant increase of total microbial biomass in limed sites (342 ng/g) compared to unlimed areas (149 ng/g). Chao1 estimates followed the same trend. But the total number of OTUs (Operational Taxonomic Units) in limed (463 OTUs) and unlimed (473 OTUs) soil samples for bacteria were similar. For fungi, OTUs were 96 and 81 for limed and unlimed soil samples, respectively. Likewise, Simpson and Shannon diversity indices revealed no significant differences between limed and unlimed sites. Bacterial and fungal groups specific to either limed or unlimed sites were identified. Five major bacterial phyla including Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, and Proteobacteria were found. The latter was the most prevalent phylum in all the samples with a relative abundance of 50%. Bradyrhizobiaceae family with 12 genera including the nitrogen fixing Bradirhizobium genus was more abundant in limed sites compared to unlimed areas. For fungi, Ascomycota was the most predominant phylum in unlimed soils (46%) while Basidiomycota phylum represented 86% of all fungi in the limed areas. CONCLUSION: Detailed analysis of the data revealed that although soil liming increases significantly the amount of microbial biomass, the level of species diversity remain statistically unchanged even though the microbial compositions of the damaged and restored sites are different. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil liming still have a significant beneficial effects on soil microbial abundance and composition > 35 years after dolomitic limestone applications.

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

Nickel and Copper Toxicity and Plant Response Mechanisms in White Birch (Betula papyrifera).

Nickel (Ni) and copper (Cu) are the most prevalent metals found in the soils in the Greater Sudbury Region (Canada) because of smelting emissions. The main objectives of the present study were to (1) determine the toxicity of nickel (Ni) and copper (Cu) at different doses in Betula papyrifera (white birch), (2) Characterize nickel resistance mechanism, and (3) assess segregating patterns for Ni and Cu resistance in B. papyrifera populations. This study revealed that B. papyrifera is resistant to Ni and Cu concentrations equivalent to the levels reported in metal-contaminated stands in the GSR. Resistant genotypes (RG) accumulate Ni in roots but not in leaves. Moderately susceptible (MSG) and susceptible genotypes (SG) show a high level of Ni translocation to leaves. Gene expression analysis showed differential regulation of genes in RG compared to MSG and SG. Analysis of segregation patterns suggests that resistance to Ni and Cu is controlled by single recessive genes.

Comprehensive Transcriptome Analysis of Response to Nickel Stress in White Birch (Betula papyrifera).

White birch (Betula papyrifera) is a dominant tree species of the Boreal Forest. Recent studies have shown that it is fairly resistant to heavy metal contamination, specifically to nickel. Knowledge of regulation of genes associated with metal resistance in higher plants is very sketchy. Availability and annotation of the dwarf birch (B. nana) enables the use of high throughout sequencing approaches to understanding responses to environmental challenges in other Betula species such as B. papyrifera. The main objectives of this study are to 1) develop and characterize the B. papyrifera transcriptome, 2) assess gene expression dynamics of B. papyrifera in response to nickel stress, and 3) describe gene function based on ontology. Nickel resistant and susceptible genotypes were selected and used for transcriptome analysis. A total of 208,058 trinity genes were identified and were assembled to 275,545 total trinity transcripts. The transcripts were mapped to protein sequences and based on best match; we annotated the B. papyrifera genes and assigned gene ontology. In total, 215,700 transcripts were annotated and were compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation (in ribosome), binding, and transporter activities. Five candidate genes associated to nickel resistance were identified. They include Glutathione S-transferase, thioredoxin family protein, putative transmembrane protein and two Nramp transporters. These genes could be useful for genetic engineering of birch trees.

Genetic and metal analyses of fragmented populations of Betula papyrifera (Marsh) in a mining reclaimed region: identification of population-diagnostic molecular marker.

White birch (Betula papyrifera) is an open pollinate species that is, dominant in the Northern Ontario after land reclamation. In fact, this species represents 65% of all trees in the region. We hypothesized that the exchange of genetic information between fragmented populations by range-wide paternal introgression is possible in wind-pollinated species such as B. papyrifera. On the other hand, the effects of heavy metal contamination from the mining activities on plant growth and population dynamics are well documented. The main objectives of this study were (1) to assess the level of genetic variation, gene flow, and population sustainability of B. papyrifera after land reclamation; and (2) to determine the level of phytoavailable metals in soil and their accumulation in trees. We found that B. papyrifera is a Ni and Zn accumulator with a translocation factor of 6.4 and 81, respectively, and an indicator of Cu and Pb. The level of polymorphic loci, Shannon index, Nei's genetic diversity, observed number of alleles, and gene flow were determined for the fragmented populations within the targeted region. The percent of polymorphic loci ranged from 28% to 56%; the gene flow was also low with a value of 0.89, and the population differentiation was very high with a value of 0.36. Two population-diagnostic ISSR markers were identified. They were cloned, sequenced, and converted to SCAR markers. Overall, the fragmented populations of B. papyrifera in Northern Ontario are genetically sustainable based on the moderate level of intrapopulation variability.

Allelic clustering and ancestry-dependent frequencies of rs6232, rs6234, and rs6235 PCSK1 SNPs in a Northern Ontario population sample.

The PCSK1 (proprotein convertase subtilisin/kexin type 1) locus encodes proprotein convertase 1/3, an endoprotease that converts prohormones and proneuropeptides to their active forms. Spontaneous loss-of-function mutations in the coding sequence of its gene have been linked to obesity in humans. Minor alleles of two common non-synonymous single-nucleotide polymorphisms (SNPs), rs6232 (T > C, N221D) and rs6235 (C > G, S690T), have been associated with increased risk of obesity in European populations. In this study, we compared the frequencies of the rs6232 and rs6234 (G > C, Q665E) SNPs in Aboriginal and Caucasian populations of Northern Ontario. The two SNPs were all relatively less frequent in Aboriginals: The minor allele frequency of the rs6232 SNP was 0.01 in Aboriginals and 0.08 in Caucasians (P < 4.10(-6)); for the rs6234 SNP, it was 0.20 and 0.32, respectively (P < 0.001). Resequencing revealed that the rs6234 SNP variation was tightly linked to that of the rs6235 SNP, as previously reported. Most interestingly, all carriers of the rs6232 SNP variation also carried the rs6234/rs6235 SNP clustered variations, but not the reverse, suggesting the former occurred later on an allele already carrying the latter. These data indicate that, in Northern Ontario Aboriginals, the triple-variant PCSK1 allele is relatively rare and might be of lesser significance for obesity risk in this population.