The previous article discussed the role of gene ontology in bioremediation. It also studied its importance in identifying new genes or proteins involved in bioremediation. Gene ontology can help in annotating and identifying important genes and proteins of organisms capable of bioremediation. Gene ontology analysis can assist in phytoremediation studies to mitigate dangerous pollutants. This is done through fast identification of appropriate genes and proteins. Moreover, gene ontology can help understand the processes and pathways associated with important bioremediation capable genes/proteins. This would help researchers in creating highly capable mutants with additional advantageous properties.
Gene ontology of Arabidopsis thaliana
This article studies gene ontology analysis of all the genes that were up or down regulated in Arabidopsis thaliana when exposed to hydrocarbons. This gene list was derived from Weisman, Alkio, & Colón-Carmona (2010). They annotated the genes from microarray data of Arabidopsis thaliana exposed to varying levels of phenanthrene. This list of genes therefore signifies those genes that are regulated by the presence of Polycyclic Aromatic Hydrocarbons (PAH). They can contribute towards tolerance and resilience of Arabidopsis thaliana against PAH pollution. Moreover, mainly those genes involved in ethylene regulation and conjugation of xenobiotics were studied. This is because these genes showed the most significant extent of upregulation. Ethylene regulation genes affect the roots length but not growth of the plant. However the genes involved in conjugation of xenobiotics helped in transporting the PAH into the cell vacuoles.
Analysis of gene ID list in PANTHER
The researcher converted Arabidopsis Genome Identifiers of the important genes in the experiment to gene IDs via NCBI Entrez search. The second step was the analysis of consequent gene ID list in PANTHER Classification System (Thomas et al. 2003). The table below shows the output.
|Arabidopsis Genome Identifiers
||Gene ID||Gene Name||Gene Symbol||PANTHER Family/Subfamily||Molecular Function|
|At1g62380||842536||1-aminocyclopropane-1-carboxylate oxidase 2||ACO2||1-aminocyclopropane-1-carboxylate oxidase 2-related||Oxidoreductase activity.|
|At2g05520||815101||Glycine-rich protein 3||GRP3||Glycine-rich protein 3 related||Not Defined.|
|At3g04720||819632||Hevein-like preproprotein||HEL||Hevein-like preproprotein|
|At3g16770||820929||Ethylene-responsive transcription factor RAP2-3||RAP2-3||Ethylene-responsive transcription factor ABR1-related||Not Defined.|
|At4g11280||826730||1-aminocyclopropane-1-carboxylate synthase 6||ACS6||1-aminocyclopropane-1-carboxylate synthase 6||Transaminase activity.|
|Conjugation of Xenobiotics|
|At1g17170||838288||Thylakoid lumenal 29 kDa protein, chloroplastic||GSTU24||Glutathione S-transferase U19-related||Not Defined.|
|At2g02390||814770||Glutathione S-transferase Z1||GSTZ1||Maleylacetoacetate isomerase||Not Defined.|
|At2g16890||816190||UDP-glycosyltransferase 90A1||UGT90A1||IP03623p-related||Transferase activity, Transferring glycosyl groups.|
|At2g43820||818986||UDP-glycosyltransferase 74F2||UGT74F2||UDP-glycosyltransferase 74F1-related||Transferase activity, Transferring glycosyl groups.|
Gene ontology output for targeted gene IDs
The table shows standardized information on gene name, symbol, family or super family and molecular function of proteins from PANTHER database. PANTHER database is a comprehensive gene ontology database in collaboration with other databases. This database collects standardized and curated gene ontology terms which can form a part of further pathway analysis.
Among the ethylene regulated genes, only the functions of two genes in 1- aminocyclopropane-1-carboxylate metabolism were present with oxidoreductase (Gene ID 842536) and transaminase activity (Gene ID 826730). Both these genes, involved in ethylene biosynthesis pathway, were found to be downregulated upon exposure to the PAH phenanthrene (Weisman, Alkio, & Colón-Carmona, 2010). Ethylene in plants are useful to influence plant growth depending upon the concentrations. According to the present study, PAH negatively interferes in the ethylene signaling pathway (Pierik et al. 2006).
Future studies for Arabidopsis thaliana
In case of genes in conjugation of xenobiotics, glutathione transferases are a large group of proteins present in Arabidopsis thaliana. These proteins tag incoming xenobiotics with glutathione for transporting into the plant cell vacuole. As the table above shows, the functions of two genes (gene ID 816190 and 818986) include transferase activity and transferring of glycosyl groups. However, for the other two genes, it is difficult to define the functions. Moreover, the database carries their gene ID . These genes are upregulated in Arabidopsis thaliana in the presence of phenanthrene. Subsequently, future studies on this organism can attempt to create mutants over-expressing these proteins. Such mutants are capable of higher rate of transfer of a wide range of xenobiotics into the cell vacuole. They also effectively remove hydrocarbons from soil.
Gene network analysis
In order to further understand the function of these up-regulated genes in Arabidopsis thaliana when exposed to phenanthrene, gene-association based network analysis was performed. Interactive online tool GeneMania integrated whole genome and microarray data formed the basis of the analysis. Data originated from multiple sources. The 9 genes involved in ethylene regulation and conjugation of xenobiotics in Arabidopsis thaliana also formed a part of the study. When examined in GeneMania, they showed multiple interactions existing within the genes as well as with other genes. Network analysis revealed related genes on basis of several parameters, as described in the figure below.
GeneMania results for Arabidopsis thaliana
The images below show the GeneMania results. They present the interaction networks of all the genes related to the selected genes in Arabidopsis thaliana. For the 5 ethylene regulation genes tested in GeneMania, 20 genes are associated with them through 206 links. Image 1 below presents the possessed 10 functions. Of these, physical interactions associated 11 genes with target genes. This includes 1-aminocyclopropane-1-carboxylate synthetases and Wall-associated receptor kinase 1. The red, pink and green coloured nodes signify 1-aminocyclopropane-1-carboxylate synthetase, ethylene metabolic processes and ethylene biosynthetic processes of the genes, respectively.
Association in other genes is as a result of co-expression, predictable functions and also common protein domains. Images 2 and 3 below show the same. Considering the extent of protein interactions, co-expression and also sharing of protein domains, the ethylene regulated genes can be said to play a central role in Arabidopsis thaliana metabolism and growth.
In case of the 4 genes in xenobiotics conjugation in Arabidopsis thaliana, gene association network analysis revealed that 838 links related 20 genes. Image 4 below shows all the links existing between 4 targeted and 20 associated genes. The red and blue coloured gene nodes signify primary functions glutathione transferase activity and glutathione binding respectively.
These linkages were mainly physical interactions, predicted associations and co-expressions (Image 5 below). Analysis of the functions of associated genes revealed 19 functions. This includes secondary metabolite catabolic and toxin catabolite processes, and also gluthione transferase activity (Image 6 below).
Future studies on Arabidopsis thaliana
There exist large scale linkages among the four targeted xenobiotics conjugation genes in Arabidopsis thaliana. They therefore influence multiple processes within the organism. Future manipulation studies of these genes need to consider these associations. It will help derive highly efficient xenobiotic transportation systems. They will be capable not only of transferring PAH but also other compounds.
Overall, this article established the important of gene ontology terms and its application in gene association studies. Efficient phytoremediation of hydrocarbons using the model organism Arabidopsis thaliana can be possible. The genes and metabolic processes associated with the up-regulated genes of ethylene regulation and xenobiotic conjugation have to be considered. Furthermore, by analyzing functions of the associated genes and processes, one can propose super-mutants that possess multiple properties like remediation of multiple compounds or complete mineralization of a pollutant.
- Pierik, R. et al., 2006. The Janus face of ethylene: growth inhibition and stimulation. Trends in Plant Science, 11(4), pp.176–183.
- Thomas, P.D. et al., 2003. PANTHER: a library of protein families and subfamilies indexed by function. Genome Research, 13, pp.2129–2141.
- Warde-Farley, D. et al., 2010. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Research, 38(2), pp.W214–W220.
- Weisman, D., Alkio, M. & Colón-Carmona, A., 2010. Transcriptional responses to polycyclic aromatic hydrocarbon-induced stress in Arabidopsis thaliana reveal the involvement of hormone and defense signaling pathways. BMC Plant Biology, 10(59). Available at: https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/1471-2229-10-59?site=bmcplantbiol.biomedcentral.com.