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

Abstract

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.

Keywords: Pinus banksiana; biological process; cellular compartment; functional genomic; molecular function; nickel and copper toxicity; resistance mechanisms.

Figure 1

Heatmap of differentially expressed genes in Pinus banksiana seedlings for (a) the copper resistant compared to nickel resistant genotypes and (b) the copper susceptible compared to nickel susceptible genotypes. Differentially expressed gene values for each pairwise comparison were based on the Log2 normalized fold change. Red data represent different levels of upregulation, whereas blue data represent different levels of downregulation. The labels Nir57, Nir30, and Nir5 represent seedlings from the nickel-resistant genotypes. The labels Cur872, Cur33, and Cur67 represent seedlings from the copper-resistant genotypes. The labels Nis15, Nis31, and Nis58 represent seedlings from the nickel-susceptible genotypes. The labels Cus42, Cus16, and Cus542 represent seedlings from the copper-susceptible genotype.

Figure 2

Volcano plot of differentially expressed genes in Pinus banksiana seedlings for (a) the copper resistance genotype compared to nickel resistance genotypes and (b) the copper susceptible genotypes compared to nickel susceptible genotypes. Brown data points represent upregulated gene expression, whereas blue data points represent downregulated gene expression relative to the susceptible genotypes. Grey points indicate no significant difference from the nickel-susceptible genotypes. LogFC is the log-fold change of the copper-susceptible genotypes relative to the nickel-susceptible genotypes. Log10(FDR) is the log10 of the false discovery rate. The barrier between the nonsignificant data points (grey) and the differentially regulated genes (orange or blue) signifies a false discovery rate of 0.05 (two-fold).

Figure 3

Percent distribution of differentially expressed genes (DEGs) for (a) biological processes for copper resistant (RG) compared to nickel resistant (RG) and (b) molecular functions for copper RG compared to nickel RG. DEGs from copper RG compared to nickel RG were annotated and allocated to terms within the biological processes category and molecular function category using Omicsbox/Blast2GO. Terms that had a total percentage of expressed genes lower than 2% were collectively categorized under the term “other.”

Figure 4

Percent distribution of the (a) top 100 upregulated genes in biological processes for copper resistant (RG) compared to nickel resistant (RG) and the (b) top 100 downregulated genes in biological processes for copper RG compared to nickel RG. The top upregulated and downregulated genes from copper RG compared to nickel RG were annotated and allocated to terms within the biological process category using Omicsbox/Blast2GO. Terms that had a total percentage of expressed genes lower than 2% were collectively categorized under the term “other.”

Figure 5

Percent distribution of the (a) top 100 upregulated genes in molecular function for copper resistant RG compared to nickel resistant (RG) and the (b) top 100 downregulated genes in molecular function for copper RG compared to nickel RG. The top upregulated and downregulated genes from copper RG compared to nickel RG were annotated and allocated to terms within the molecular function category using Omicsbox/Blast2GO. Terms that had a total percentage of expressed genes lower than 2% were collectively categorized under the term “other.”

Figure 6

Percent distribution of the (a) top 100 upregulated genes in cellular compartment location for copper resistant (RG) in comparison to nickel resistant (RG) and the (b) top 100 downregulated genes in cellular compartment location for copper RG in comparison to nickel RG. The top upregulated and downregulated genes from copper RG compared to nickel RG were annotated and allocated to terms within the cellular compartment category using Omicsbox/Blast2GO. Terms that had a total percentage of expressed genes lower than 2% were collectively categorized under the term “other.”

Figure 6

Percent distribution of the (a) top 100 upregulated genes in cellular compartment location for copper resistant (RG) in comparison to nickel resistant (RG) and the (b) top 100 downregulated genes in cellular compartment location for copper RG in comparison to nickel RG. The top upregulated and downregulated genes from copper RG compared to nickel RG were annotated and allocated to terms within the cellular compartment category using Omicsbox/Blast2GO. Terms that had a total percentage of expressed genes lower than 2% were collectively categorized under the term “other.”