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INVESTIGATOR: Ren, S.

PERFORMING INSTITUTION:
VIRGINIA STATE UNIVERSITY
PETERSBURG, VIRGINIA 23803

STRENGTHENING VSU`S INTERDISCIPLINARY RESEARCH CAPACITY TO ADDRESS HEAVY METAL CONTAMINATIONS IN VIRGINIA URBAN SOILS

NON-TECHNICAL SUMMARY: Urban Agriculture (UA) is growing rapidlyacross the United States due to the increasing demands of locally grown produce and heightened health perspective. UA offers a range of health, social, environmental and economic benefits, however, it also presents its own challenges/concerns. One of the major concerns is the potential soil contaminations by heavy metals in urban areas. Such concern has also been raised by local stakeholders at Virginia UA Summit in 2015. In response to stakeholders' concerns, we are buildingan integrated interdisciplinary team has been established atVirginia State University (VSU) to address the potential urban land contamination and the related safety concerns. Previous research in PI's Laboratory, funded by USDA Evans Allen program, identified purslane as a significant potential accumulator of various heavy metals, such as arsenic, lead and chromium, in its above-ground tissues.One of purslane accessions not only can accumulate hexavalent chromium (Cr6+) in its shoots, but also that Cr6+ promotes its growith. Such characteristics makepurslane a potential plant species for phytoremediation of soil/water Cr6+ contaminations. In addition, the PI found out that there is variation among purslane biotypes in their ability to accumulate heavy metals. Building up on these findings, this projectwill test the potential use of purslane to clean up soils contaminated by arsenic, lead and chromium and determine the mechanisms of chromium accumulation through comparative genomics study. The PI willalso screenselected fruits and vegetables for heavy metal accumulation in their edible parts and identify species and/or varieties that are safe to grow and consume on mild contaminated urban soils.

OBJECTIVES: The goal of this proposal is to strengthen VSU's capacity for long-term research and extension service on heavy metal contaminations in UA and positions VSU to a leading role in support of urban farmers in Virginia. Three specific objectives will be investigated during the grant period, infor 1) evaluate the potential use of Purslane to clean up soils contaminated by arsenic, lead, and chromium; 2) isolate possible candidate genes responsible for chromium shoot accumulation using comparative RNA-seq approach; and 3) examine selected fruits and vegetables crops for heavy metal accumulation in the edible parts and identify species and/or varieties that are safe to grow and consume on contaminated soils.

APPROACH: To implement objective 1, we will first conduct simulation experiments in the greenhouse to test purslane's ability for bioremediation of As, Pb and Cr6+. The greenhouse results willserve as a guide for subsequentfield experiments. For field experiments, four selected purslane accessions will be grown at twosites with known heavy metal contamination levels. The above-ground biomass will be harvested for heavy metal analysis. Experiments will follow randomized complete block design (RCBD) with three replicates, and the collected data will be statistically analyzed using SAS package. To implement objective 2, purslane accessions GT, TJ and GG will be grown in greenhouse and treated with 300ppm Cr6+. Shoots and roots will be collected separately at 0, 2 and 72 hours post of treatment (POT). Total RNA will be extracted and subject for cDNA library construction and RNA-seq analysis. RNA-seq data will beanalyzed using software freely available on CyVerse (www.cyvers.org), federal funded cyber infrastructure. RNA-seq data will be validated through RT-PCR and candidate genes will be further examined for their expression patterns under normal and Cr6+ treated conditions. The selected candidate genes will be served as first step to engineer plant species for Cr6+ accumulation. To implement objective 3, commonly grown urban crop seeds consisting root, shoot, leaf and edible fruits, such as carrot, sweet potato, kale, collard green and tomato, will be purchased from different sources (different varieties) and tested in greenhouse for their absorption and accumulation of As, Pb, and Cr at their edible parts. At least five varieties for each species will be tested. Field experiments will also be conducted for all selected species and/or varieties. Such information is essential for the determination of which species or variety within species have potential to safely grow on contaminated soils and yet still be safe for human consumption.

PROGRESS: 2020/03 TO 2021/02
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this grant period, we have a Postdoctoral research associate being recurited on this project for about 5 months and trained for scientific mewthodoligy related to the project. 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?Detail analysis of the trasnscriptome will be the major target for next reporting period, Eventually we will identify some promising genes that are important for Cr tolerance in crop species. In addition, we will test Cr and Pb accumulation in two different rooty vegetables (radish and beet) with 4 varieties each species included.

IMPACT: 2020/03 TO 2021/02
What was accomplished under these goals? During this grant period, we donducted transcriptome analysis to identify the expression profiles in response to chromium treatment. Two different puralsne accessions, one with chromium sensitive and the other chromium tolerance were selected and subjected to a 24 hour 200ppm Cr6+ treatment (with water as control treatment). Roots and shoots were collected seperately after treatment completed. All treatments and tissue collections were biologivcally triplicated. Total RNAs were isolated and subjected for RNA-seq analysis. Overall, 1043 millions clean reads were obtained from all samples and at least 40M clean reads were received from each sample. After de novo analysis, at least 71% of clean reads from each library were mapped to existing genes in all available database. With a log 2 fold as significantl cut off, we analysed expression difference between Cr6+ treated and control samples for both Cr tolerant and sensitive accessions. For accession GT, a Cr tolerance purslane, significant amount of genes were altered in their expression in root after Cr6+ treratment, where more than 10000 gene clusters were upregulated and about 9000 gene clusters were down regulated by Cr6+ treatment. Much less gene clusters were affected by Cr6+ treatment in their shoot tissues with only 2746 gene clusters up and 2049 gene cluster down regulated by Cr6+ treatment. Similar trends were observed for accession EG, a Cr sensitive purslane accession, with about 7000 gene clusters up and 7000 down in root tissues and about 4000 up and 2700 down regulated in shoot tissues in response to Cr6+ treatment. GO enrichment analysis were conducted to identify the pathways that is related to the Cr6+ treatment or the response to the treatment. In general, kinase activity, transmembrane transport activity and ion binding activity are the most affected by Cr6+ treatment in both root and shoot tissues. For GT accession, Kinase activity and ion binding activity were significantly uregulated, while transmembrane transport activity is down regulated by Cr6+ treatment in roots. However, in shoot tissues, exact opposite was fund with kinase activity and ion binding activity were down regulated and transport activity upregulated. In addition, response to stress was all down regulated in both root and shoot tissues indicating that Cr6+ treatment for GT accession does not consider as a stress for Cr tolerant accession. In consistance, photosynthesis pathway genes were significantly upregulated in shoots by Cr6+ treatment. This finding supports our observation that after Cr6+ treatment, accession GT grew better and even doubled biomass comparing to untreated control. For EG accession, where it is sensitive to Cr6+ treatment, kinase activity and ion binding activity were significantly up regulated by Cr6+ treatment in both root and shoot tissues, but transmembrane transport activity were significantly down regulated by Cr6+ treatment in roots and shoots. Such response difference between Cr tolerant and Cr sensitive purslane accessions to Cr6+ treatment may suggest a molecular mechanism in control of Cr6+ transport. Furthermore, in consistant with the sensitivity to Cr6+, genes responding to stress were significantly up regulated in EG shoots upon Cr6+ treatment, and genes controlling photosynthesis were drastically downregulated by Cr6+ treratment. This transcriptome analysis suggests two potential molecular explainations on Cr tolerance in GT accession: 1) although Cr6+ is toxic to plants, upon uptaken into GT roots, it may be converted into Cr3+ where Cr3+ is essential element for plants (as well as animal) growth, therefore it is not only toxic but even promote its growing as we demonstrated previously; 2) when taking up Cr6+ and transporting from roots to shoots, the activated transmembrane transporters in shoots will transport Cr6+ into vacule and stored it there to avoid causing damage to the plants. However, considering our previous findings where Cr6+ treatment significantly promotes its biomass growing, we hypotheses that the first explanation could be the case and is warranting for further invistigation.

PUBLICATIONS (not previously reported): 2020/03 TO 2021/02
No publications reported this period.