Progress 09/01/12 to 08/31/15
Outputs
Target Audience:The results of this mine-site research focus towards coal mine companies, ecological genomicists, weed control specialists, those involved in reclamation, and students and researchers participating in phytoremediation. The analysis provided will also be useful for the WV DEP (Department of Environmental Protection) and other state regulatory agencies in the Appalachian region. Graduate and undergraduate students in biotechnology and biology at West Virginia State University directly benefitted through the education and training experiences that were provided. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The grant provided professional development training for three graduate students who worked on the plant transcriptome project. Two undergraduate students were also involved in the plant gene expression study and the soil microbiome study. In addition, one post-doctoral scientist and two technicians contributed to the research. The PD (Huber) used the experience and knowledge gained from the project to enhance his classes, Environmental Microbiology (BIOL 460/660) and Current Concepts in Biotechnology (BT567). How have the results been disseminated to communities of interest?Our research has been publically disseminated and is available to a wide audience, including private companies, students, and researchers working on heavy metal remediation and environmental reclamation. To date, we have presented our results in the following two publications. Additional publications will occur during the next year. Saminathan T,, Malkaram SA, Patel D, Taylor K, Hass A, Nimmakayala P, Huber DH, and Umesh K. Reddy.2015.Transcriptome Analysis of Invasive Plants in Response to Mineral Toxicity of Reclaimed Coal-Mine Soil in the Appalachian Region. Environmental Science & Technology.DOI: 10.1021/acs.est.5b01901. Thangasamy Saminathan, Sridhar A. Malkaram, David H. Huber, Dharmesh Patel, Amir Hass, Padma Nimmakayala and Umesh K. Reddy. 2014 March 6-8. Genomic Approaches for Appalachian Coal Mine-site Reclamation. Poster presented at:MCBIOS XI. Annual Bioinformatics & Computational Biology Conference;Stillwater, OK, USA. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objectives: Application of functional genomics and genome wide association studies to identify genes and favorable combinations of alleles with reference to coal mine site toxicity; measurement of microbiome ecosystem processes across a mine site reclamation gradient; measurement of the recovery of soil properties on reclaimed mine sites. Comparative transcriptome analysis of mine-site invasive plants: We have grown invasive plants, mugwort and goutweed, in two mine-site soils (35 years as MS-I; 10 years as MS-II of post-mining) and in control growth medium Promix. Mineral content analysis as well as gene expression analysis of the plants was performed. We ended up with the following results. MS-I and MS-II had high levels of plant available Al, Fe, and Mn. Available Ca, K, Mg, Na, P, and S levels were low in both mine soils. The pH range was higher with Promix than MS-I and MS-II (∼5.8 to 6.2 vs 4.4 and 4.8, respectively). Nitrogen in both mine soils was close to 1 ppm as compared with 90 ppm in Promix. The low soil nutrient content in mine soils led to stunted growth and symptoms of P-deficiency in both plants. In our acidic soil conditions, high Al tolerance and hyperaccumulation in mugwort might be mediated by Nramp aluminum transporter (NRAT1), malate transporter (ALMT1), and/or ABC transporter. From our transcriptome data, we found several commonalities between two plants as well as for two mine sites. Some of the genes were upregulated or downregulated in both plants when compared with the control. These genes constitute only a small fraction of the full set of differentially expressed genes. The variation in transcriptome between plants was much higher than between the mine sites. Certain key gene regulators such as nitrate, ABC and Al transporters, TFs, cytochrome p450, and glutathione s-transferase were up- or downregulated in MS-I or MS-II. More importantly, genes such as nitrate transporter, cytochrome p450, and rhicadhesin receptor were up- or downregulated with both mine-site soils, for both species. Both plants accumulated high Al and Fe content, and concomitantly the genes for ABC transporter and ZIP cation transporter were expressed in both species, for hyperaccumulation of these elements. Together, despite increased number of genes/transcripts in goutweed, the results indicate that increased accumulation of metals in mugwort might result from a greater number of genes/ transcripts in the transporter category in mugwort than goutweed. These genes/transcripts are transporters, which play key roles in sequestering heavy metals into vacuoles without having detrimental effects on physiological homeostasis. Al has been identified in xylem sap as Al-citrate in several Al hyperaccumulators. In addition, antioxidants directly or indirectly protect cells against adverse effects of heavy metals by increased distribution of ROS in vacuoles and chloroplasts. The findings from our transcriptomics will be useful for restoration/reclamation and for phytoremediation of contaminated soils and water. Characterization of the soil microbiome and critical soil properties across a mine-site reclamation gradient: Soil microbiome recovery and soil chemistry were measured across a mine site reclamation gradient that spanned one to 34 years following reclamation. Sampling was conducted to assess whether soil microbiome functions (ecosystem processes) and essential soil properties were recovering through time. Five reclamation sites were identified and sampled as well as one control (forest) site. Five meter square quadrants were set-up in each location, the vegetation cover in each site was measured, and soil samples were collected for chemical and microbiome analyses. From the six sample sites, 18 metagenomic DNA samples were collected and sequenced with Illumina HiSeq technology. More than 60 gigabases of DNA were generated. The comparative analysis of the microbiomes is currently underway using standard technology such as MG-RAST. Plant community structure recovery was also characterized in the same locations. The five different reclaimed mine sites showed positive correlation (R2 = 0.81) between time since reclamation and accumulation of soil organic matter. At the same time period, soil pH decreased from above natural value at perturbation (7.33) to 4.33 at 34 years after reclamation, below that of nearby native mixed hardwood forest (4.77). Soil cation exchange capacity (CEC) increased from 5.8 meq 100g-1 at one year after reclamation to an estimated 9.1 meq 100g-1 after 34 years. The increase in CEC was linearly correlated with soil organic matter content (y = 0.7867x + 4.1668; R2 = 0.93). While Base Saturation was also highly correlated with time (R2 = 0.97), it exponentially decreased from 99% one year after reclamation to 35% at the site reclaimed 34 years prior to sampling. The results of this study suggest that soil organic matter play a dominant role in soil forming processes in these sites. Buildup of soil organic matter to levels similar to that of native hardwood forest is estimated to occur after 45 years of reclamation. The contribution of mineral phase to soil forming processes seemed restricted, likely due to relatively slow kinetics of mineral and mineralogical processes, leading to limited resilience of the developed soil to induced processes by organic matter decomposition and impact of organic acids. Noteworthy, a high time-dependency was found among the different soil parameters tested and time since reclamation. This is in spite of the fact that the different sites represent different reclamation practices and vegetation types, as they denote the changes and development of reclamation practices as they occurred over the 34 year period (from 1980 to 2013).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Saminathan T,, Malkaram SA, Patel D, Taylor K, Hass A, Nimmakayala P, Huber DH, and Umesh K. Reddy.2015.Transcriptome Analysis of Invasive Plants in Response to Mineral Toxicity of Reclaimed Coal-Mine Soil in the Appalachian Region. Environmental Science & Technology.DOI: 10.1021/acs.est.5b01901.
|
Progress 09/01/13 to 08/31/14
Outputs
Target Audience: The target audiences are ecologists and private companies working on mine-site reclamation, and scientists working on plant genomics and soil microbial ecology. Undergraduate and graduate students will also gain technical training in plant and microbial genomics, soil chemistry, and soil microbiology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students worked in the project to assist RNA isolation and preparation for sequencing. Currently another graduate student is helping to set up screening plants in water collected from mine-sites. An undergraduate was involved setting up experiment on gene expression differences among various mine-site reclaimed soils. Two technicians have also contributed to the research: one full-time and one part-time. How have the results been disseminated to communities of interest? The results have been disseminated by presenting a poster at the conference MCBIOS XI, Annual Bioinformatics & Computational Biology Conference; 2014 March 06-08; Stillwater, OK, USA. What do you plan to do during the next reporting period to accomplish the goals? Plant genomics analysis will be completed. Soil chemistry and soil microbiome analysis will be completed.
Impacts
What was accomplished under these goals?
In this study, we aim to identify gene regulation during high and low nutritional availability by using two different plant species. We are using a holistic approach rather than evaluating one component at a time in order to understand the overall impact of mine-site soil on plant growth. We are also evaluating whether current mine site reclamation strategies promote the recovery of those soil ecosystem services that are provided by the soil microbiome. Soil properties and elemental analysis: A mine-soil pH range of 6.0 to 7.5 is ideal for forages and other agronomic uses. However, mine-site soil has pH between 4.4 and 4.8. A mine-soil pH that is too high in acidic also inhibits growth due to micronutrient deficiencies. In addition to low pH, mine-site soil possesses low N, P and K along with high Ca, Mg, Fe, Zn, Mn and Cu compared to Pro-Mix. When plants are grown under abnormal nutrient conditions for prolonged time, plants start to absorb and accumulate in their leaf, stem and other aerial parts. Using inductively coupled plasma (ICP) analysis, our preliminary results show that hyper accumulation of Al, Fe, K and Mn occurs in mine-site I soil. Aegopodium exhibits hyper accumulation of Fe followed by Al in mine-site I. Comparing both mine-sites, mine-site I showed extreme accumulation of elements. Transcriptome analysis of Aegopodium and Artemisia: When plants are grown in adverse conditions, element-specific gene regulations occurs in order to respond to the condition. There are two types of responses; a) exclusion of elements from the roots and b) allowing elements (inclusion) and tolerating. To perform this hyper accumulation or exclusion, plants do have physiological system along with differential regulation of gene expression. The transcriptome data from RNA-seq reveals that umpteen number of genes both up- and downregulated in both the plants in response to mine-site soils Figure 2 (top 1000 genes). These genes were further narrowed-down based on up- and downregulation in both sites. Most of them element-related regulators such as metal transporters, receptor kinases, chelating proteins, glutathione S-transferase, ferric reductase, channel proteins, Fe transport protein, copper transporter, PHO1-like protein (for phosphate), and transcription factors. They were up- or downregulated irrespective of element concentration in the mine-site soils depends on the exclusion or inclusion mechanisms. The list of up- and downregulated genes from both mine-site experiments was validated with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). From our results, few genes were upregulated in both sites indicating that the two minesites possess similar physicochemical properties at least for few metals/elements if not all. Soil microbiome analysis across a reclamation gradient: Sampling was conducted to assess whether soil microbiome functions (ecosystem processes) were recovering across a surface mine site reclamation gradient. Five sites were identified and sampled that have been subjected to surface mining and reclamation representing 1 to 20 years post mining. Ten meter square quadrants were set-up in each location, the vegetation cover in each site was recorded, and soil samples were collected for chemical analyses and microbiome analysis (metagenomic sequencing). Chemical analysis using ICP technology and DNA sequencing are currently in progress.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
The research was presented at the following meeting:
Thangasamy Saminathan*, Sridhar A. Malkaram, David Huber, Dharmesh Patel, Amir Hass, Padma Nimmakayala and Umesh K. Reddy. Genomic Approaches for Appalachian Coal Mine-site Reclamation. Poster presented at: MCBIOS XI. Annual Bioinformatics & Computational Biology Conference; 2014 March 06-08; Stillwater, OK, USA.
|
Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The target audience is the Appalachian coal mining industry, environmental scientists and ecologists interested in land reclamation, and graduate and undergraduate students in the biology and biotechnology programs at West Virginia State University. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A postdoctoral associate was recruited for carrying out various experiments involved. Two undergraduate students worked in the project and got trained in RNAseq and function genomics. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? The plant genomic characterization of traits that are associated with growth on mine site soils will be continued. The soil microbiome will be sampled from reclaimed soils of varying age. The natural vegetation cover and soil chemical properties on these sites will be documented.
Impacts
What was accomplished under these goals?
Minesite samples up to 20 cm depth were collected from two different locations. Dried and sieved samples were used to perform chemical analysis including pH, soluble salts, and extractable nutrients (phosphorus, calcium, magnesium, potassium). Rest of the samples was used to evaluate plant growth involving two invasive species. Rhizomes and stolons of Mugwort (Artemisia vulgaris) and gout weed (Aegopodium podagraria) respectively were used to grow in pro-mix (Premier Tech Ltd) as well as mine-site samples in two replications. Roots of 30 day old plants were collected from all the replications for RNA isolation. Samples were paired-end sequenced to produce a minimum 25million reads/sample using Illumina HiSeq2500 system. In addition to low pH (4.4 and 4.8), chemical analysis of soil samples revealed that mine-site samples contained low N, P and K and high Ca, Mg, Fe, Zn, Mn and Cu, when compared to pro-mix. Our results from Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis indicated hyper accumulation of Al, Fe, K and Mn in the leaves of Artemisia and Aegopodium grown in the mine-site samples. RNAseq results showed 54944 and 63864 transcripts assembled for Artemisia and Aegopodium respectively. Among these, 1328 and 3547 were significantly differentially regulated in mine-site samples when compared to pro-mix in Artemisia and Aegopodium respectively. For Artemisia, in comparison with pro-mix, 157 transcripts were upregulated when compared to 1171, which were downregulated. In the case of Aegopodium , 1426 and 2121 transcripts were up and down regulated respectively in comparison with pro-mix. 1-aminocyclopropane-1-carboxylate oxidase, 2-oxoglutarate-dependent dioxygenase, early nodulin-like protein 1-like, GDSL esterase lipase, glutathione s-transferase, high affinity nitrate transporter histone H2a, 6-phosphogluconate dehydrogenase and ankyrin repeat-containing protein were important among the upregulated. Important pathways that showed upregulation were ABC transporters, alanine aspartate and glutamate metabolism, arginine and proline metabolism and ascorbate and aldarate metabolism among the plants grown in mine-site samples. DnaJ heat shock n-terminal domain-containing protein, lipoxygenase, WRKY transcription factor and heat shock protein 70 were among highly downregulated genes in the plants that were grown in mine-site samples. Among the important pathways that showed significant downregulation were apoptosis signaling, cysteine and methionine metabolism and endocytosis. We noted a comparable trend of differential expression of several transcripts among Artemisia and Aegopodium grown in mine-site samples. In addition, the response noted from the ICP-MS analysis is similar across the plant species in the current research.
Publications
|