Source: SOUTHERN UNIV submitted to NRP
GENETIC APPROACH TO ASSESSING UV-B RADIATION TOLERANCE CAPACITY IN SELECTED SOUTHERN BROADLEAF TREES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1030357
Grant No.
2023-38821-39967
Cumulative Award Amt.
$300,000.00
Proposal No.
2022-09970
Multistate No.
(N/A)
Project Start Date
Apr 15, 2023
Project End Date
Apr 14, 2026
Grant Year
2023
Program Code
[EQ]- Research Project
Recipient Organization
SOUTHERN UNIV
(N/A)
BATON ROUGE,LA 70813
Performing Department
(N/A)
Non Technical Summary
Ozone depletion has resulted in a significant increase in solar ultraviolet radiation (UV-B, 280-315nm and UV-A, 315-400nm) on the earth's surface. Relatively little information exists on the impact of UV radiation on forest tree species, which account for 80% of the global net primary production. With the future uncertainty of stratospheric ozone recovery and global climate change, it is critical to evaluate the impact of solar UV exposure on trees. Scientists at Southern University in collaboration with USDA have assessed more than 30 southern broadleaf tree species and gained good understanding of their biophysical, biochemical, and anatomical aspects of the UV tolerance mechanism during last two decades. With the existing knowledge we propose to investigate their genetic aspect of UV tolerance mechanism. Specifically, we will investigate UV induced two of the most abundant mutagenic and cytotoxic DNA lesions (CPD and 6-4 photoproducts), a general marker of DNA oxidative damage (promutagenic base 8-oxo-2'-deoxyguanosine), and protein and gene expression of UV-specific DNA polymerase during leaf emergence, mature and senescence in a growing season. The goal is to assess the seasonal UV (A/B) induced genetic changes, DNA damage and repair mechanism in selected southern trees. The project will lead to new knowledge discoveries that will shape our comprehensive understanding of the UV tolerance mechanism in selected broadleaf trees, which will have implications in modeling and predicting forest health and designing effective forest management strategies. The project will strengthen our institutional research competitiveness and enhance research and training capabilities at Southern University.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010621108070%
1240621107020%
1320430207010%
Knowledge Area
132 - Weather and Climate; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 124 - Urban Forestry;

Subject Of Investigation
0621 - Broadleaf forests of the South; 0430 - Climate;

Field Of Science
2070 - Meteorology and climatology; 1070 - Ecology; 1080 - Genetics;
Goals / Objectives
The overarching goal of the project is to discover seasonal effect of UV(A/B) on DNA damage/repair capability in select group of major southern broadleaf tree species from genera of Magnolia, Acer, Carya, Fraxinus, Liquidamber, Populus, Quercus, Sapium, Ulmus, etc. and toassess their UV Tolerance ability. The specific Objectives are:(1) To determine UV (B/A) induced DNA damage by measuring the occurrence of 6, 4-photoproducts of thymine and cytosine (6,4PPs) and the cyclobutane pyrimidine dimers (CPDs) at different leaf developmental stages (emergence, mature and senescence) in selected trees as they are continuously exposed to solar UV (B/A) radiation throughout a growing season.(2) To determine a general marker of DNA oxidative damage by measuring the premutagenic base 8-oxo-2'-deoxyguanosine (8-oxodG) at the different stages of the leaf development in the selected trees during a growing season.(3) To determine the repair of UV (B/A) induced DNA damage by measuring protein and gene expression of UV-specific DNA polymerase and CPD- and 6,4PP-photolyase.(4) To assess genetic aspect of UV tolerance relative to DNA damage and repair mechanism based on the above three objectives.
Project Methods
Leaf Sample Collection: Leaves of the selected southern tree species from genera of Magnolia, Acer, Carya, Fraxinus, Liquidamber, Populus, Quercus, Sapium, Ulmus, etc. will be collected from individual trees growing within the city of Baton Rouge, Louisiana at three stages during a growing season: the leaf emergence stage (March/April), mature stage (July/August), and the leaf senescence stage (October/November) for all the deciduous trees. At each sampling stage, 20 to 30 leaves will be sampled from the sun portion of an unshaded individual tree at the terminal 20-50 cm of a branch or foliage. Three trees will be sampled per species. Samples will be collected at approximately the same time during each sampling day (clear-sky days) between 8-10 am and placed in a humidified plastic bag in an insulated box for transporting to the laboratory and stored in -80 oC until analysis. The experimental design is a split plot arrangement (in time) in a completely randomized design with three replications. The main plots are individual trees, and the sub plots are leaf growth stages. The experiment will be repeated for three growing seasons. The data will be analyzed as a repeated measures experiment.Meteorological Data: Monthly precipitation total and temperature data (maximum, minimum, and average) of the experimental site (the city of Baton Rouge, Louisiana) will be obtained through the National Oceanic and Atmospheric Administration (NOAA) - National Climate Data Center (NCDC) National Weather Service from the Baton Rouge Metro Airport weathering monitoring station. The cumulative daily, monthly, seasonally, and yearly UV-A, UV-B radiation totals for the experimental site will be obtained through the USDA-UV-B Monitoring and Research Program from its Baton Rouge UV monitoring station (Benhur Farm, 10 miles from our campus). The DNA damage induced by UV (B/A) radiation will be measured based on the occurrence of 6,4-photoproducts of thymine and cytosine (6,4PPs) and the cyclobutene pyrimidine dimers (CPDs). A general marker of DNA oxidation such as 8-oxo-2'-deoxyguanosine (8-oxodG) will be determined along with 6,4PPs and CPDs to find out the status of oxidative stress in these plant materials. The DNA repair of UV (B/A)-induced DNA damage will then be determined through measurement of protein and gene expression of UV-specific DNA polymerase and CPD- and 6,4PP-photolyase. The analytical steps are summaried as follows. Isolation of DNA: a commercially available DNA isolation kit (DNeasy Plant Kit; Qiagen) will be used. The leaf tissues will be ground to a fine powder in liquid N2 and thereafter the manufacture suggested procedures will be followed. The leaf tissue will be dispersed in 5 mL of the modified AP1 buffer (Qiagen) supplemented with metal-chelating agents (5 mM deferoxamine and 20 mM EDTA) and ground in a mortar. RNase A will be added to the mixture, and the solution incubated at 65 °C for 10 min. Remaining steps will be exactly followed as per manufactures instructions. In both the case DNA dissolved in RNase-free water or Tris-EDTA (TE) buffer will be subjected to spectrophotometric measurement at 260 nm and 280 nm to assess the quality as well as yield. The purity will be further assessed by electrophoresis on 0.9% agarose gel using Tris-acetate-EDTA (TAE) buffer.Digestion of DNA: For assays involving the estimation of 8-oxodG and 2'-deoxyguanosine (dG), DNA samples will be digested to the nucleoside level in acetate buffer, pH 5.0 using a combination of DNase II (2 units/μg of DNA), phosphodiesterase II (0.74 units/mg DNA), and type II acid phosphatase from white potato (3 units/mg DNA) for 2 h as described by Marsch et al. (2004).Determination CPDs and 6,4PPs: Cyclobutene pyridinium dimers will be determined by ELISA using monoclonal antibodies TDM-2 and 6,4M (Cosmo Bio; Carlsbad, CA) that selectively recognize CPDs and 6,4PPs, respectively (Roza et al., 1988; Morit et al., 1991; Sinha et al., 2001; and Perdiz et al., 2000). The values will be expressed as number of CPDs/Mbp or number of 6,4PPs/Kbp.Measurement of oxo8dG: 8-Oxo-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) in DNA samples will be resolved by reversed- HPLC and quantified by electrochemical detection as described by Floyd et al. (1990), Uppu et al. (1996), Marsch et al., 2004. The identity of 2dG and oxo8dG in plant tissue samples will be determined by co-elution with authentic standards as well as H-NMR. The data will be expressed as mol of oxo8dG/105 mol of 2dG present in the DNA.Expression of CPD-Photolyase (CsPHR) and UV DNA-Polymerase: The levels of CsPHR transcripts will be estimated by quantitative RT-PCR as well as the CPD- photo repair activity. Total RNA will be isolated and purified from leaves using a RNeasy Plant RNA isolation kit (Qiagen). The yield RNA and its purity will be determined spectrophotometrically. Approximately 1 μg of RNA will be reverse transcribed using a cDNA synthesis kit (Qiagen). The cDNAs will be subjected to RT-PCR analysis using custom made primers specific for CPD-photolyase (CsPHR), UV DNA-polymerase, and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The cDNA containing master mix will be prepared according to the manufacture's instruction and the RT-PCR analysis performed ABI 7500 PCR apparatus and the fold change in gene expression will be calculated based on Kumar et al. 2009, 2010. The fold-increase in the expression of genes will be calculated with reference to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.Assay of Endonuclease-V: Leaf tissues (100 mg each) will be ground to a powder in liquid nitrogen and extracted in 1 mL of buffer containing 0.1 M Tris-HCl (pH 7.5), 1 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM phenylmethyl sulfonyl fluoride, 5 mM phenanthroline, 0.1% Triton X-100 and 3% polyvinylpyrrolidone. The samples will be centrifuged at 4°C for 10 min at 16000x g and the supernatant used as enzyme source. Endonuclease-V activity will be determined in 30 μL reaction volume containing 0.2 pg crude protein, 50 mM EDTA (pH 8.0), 20 mM Tris-HCl (pH 7.2) and 0.2 μg of UV-C-irradiated DNA (form I Ml 3mp 19 DNA: - 30 CPD per molecule). Reactions will be incubated for 30 min at 37 °C and stopped by addition of proteinase K (50 units/mL) and gel loading buffer. The samples will be heated at 70 °C for 10 min, electrophoresed through 0.9% agarose in Tris-acetate-EDTA (TAE) buffer containing 50 pg/mL ethidium bromide. The DNA fluorescence will be recorded using a gel documentation system.DNA-Photolyase Activity: Photolyase activity will be assayed based on the method of Hada et al. (2000).ELISA using CPD-specific monoclonal antibody (XP-01): DNA recovered from the photolyase reaction (see above) will be dissolved in 1 mL of 10 mM PBS, pH 7.4 and denatured in boiling water for 10 min. The denatured DNA (50 μL) will be added to micro titer well plates and allowed to dry at 90 °C for 1 h. The monoclonal antibody against CPD-specific XP-01, TDM-2 or 64 M-2 will be utilized to perform the ELISA as described earlier.Data analysis: All data will be analyzed as repeated measures in a completely randomized design with three replications for each variable. The main plots are individual trees, and the sub-plots are leaf growth stages. The experiment will be repeated for three growing seasons (2023, 2024, 2025 three growing seasons). The data will be analyzed by statistical package using R or SASsoftware.

Progress 04/15/23 to 04/14/24

Outputs
Target Audience:The target audience includes the UV monitoring/research community and the generalpublic. The resulting information on how major Southern tree species in the USA interact with UVB radiationwill be helpful not only to the scientific community to understand the UVB-induced DNA damage and repair mechanism, but also to forestry and natural resources practitioners in selectingUV-tolerant tree species for planting and forest regeneration. The project also targets training next-generation scientists and forestry professionals by providing research and experientiallearning opportunities to postdocs, and graduateand undergraduate students. Changes/Problems:Our university has been renovatingour research building since 2023, as a result,our research lab and research equipment are being relocated to different areas, which has delayed our research progress. However, since we hope we willhave the opportunity to request for no-cost extension for 1 more year or up to 2 more years, we believe we will be able to accomplish the goal and objectives set for this project in time. What opportunities for training and professional development has the project provided?The project has generated opportunitiesfor the project director and co-directors, a postdoc research associate, and 3 graduate students to participate in research activities,receive professional training, attend conferences, and present papers. The research collaboration has enabled twostudent internships with USDA ARS Honey Bee Lab to receive research-related training. Professional development includes attending conferences such as the the 2nd World Forum on Urban Forests organized by FAO and USDA-FS in 2023, and the 21st BiennialARD (Association of 1890 Research Directors, Inc.) Symposium in 2024. The project also provided various opportunitiesto expose undergraduate students to learn about the USDA UVB monitoring and research program and UVB's impacts on the ecosystem. How have the results been disseminated to communities of interest?The PI, Dr. Yadong Qi was invited by the USDA UV-B Monitoring and Research Program and the United Nations Environment Program Environmental Effects Assessment Panel to give a presentation entitled "Assessing UV Tolerance in Broadleaf Trees and Modeling Urban Tree Canopy Interception of Solar UV Radiation" at the Monitoring, Modeling, and Assessing the Environmental Effects and Changes in Solar UV Radiation and Climate Workshop on Sept 18, 2023 in Colorado State University, Fort Collins, CO. The presentation generated a high level of interest in how forests interact with UVB radiation. Dr. Yadong Qi, the PI along with her team was selected to give an oral presentation on "Urban Tree Canopy Reduction of Solar Ultraviolet Radiation: Mechanism and Assessment" at the 2nd World Form on Urban Forests organized by the United Nations FAO and USDA-FS inWashington DC, on Oct. 16,2023, https://www.worldforumonurbanforests.org/past-events/wfuf-washington-2023.htmlThe presentation was well-received by conference attendees, urban forestry practitioners, and the scientific community. In addition, the project personnel and graduate students made two presentations at the 21st BiennialARD Symposium inNashville, Tennessee, one presentation was on "Quantifying Urban Forest Benefits on Reduction of Solar UV-A and UV-B Radiation" and the other on "Localization of UV-Absorbing Compounds in Green Ash (Fraxinus Pennsylvania) and Nuttall Oak (Quercus nuttallii) Trees in the Temperate Deciduous Forest Biome". A graduate student (L. Dasari) made a presentation on "Quantifying the Effects of Tree Canopy on Reduction of Air temperature in Urban Environment" at the2ndSouthern University Graduate Student Research Symposiumon April 24, 2023, in Baton Rouge, LA. The PI Dr. Qi was invited to give an online talk entitled "Assessment of Urban Tree Canopy Reduction of Solar UV Radiation and Mechanism of UV Tolerance in Broadleaf Trees" at the 8th China Forestry Science Conference - Urban Forestry Division onJuly 29, 2023. What do you plan to do during the next reporting period to accomplish the goals?We plan to set up thenewly acquiredHigh-PerformanceLiquid Chromatograph and Mass Spectrometer (HPLC/MS) system and develop a protocol toanalyzethegeneral marker of DNA oxidative damage by measuring the premutagenic base 8-oxo-2'-deoxyguanosine (8-oxodG) at the different stages of the leaf development in the selected trees during a growing season.We will continue to work with our collaborators including the USDA-UVB Monitoring and Research Program at Colorado and the USDA-ARS-Honey-Bee Lab to strengthen our research training and data analysis. We will work with Novegene USA and identify other institutions/entities to explore the means for successfully measuring the protein and gene expression of UV-specific DNA polymerase and CPD- and 6,4PP-photolyase,which will help us better understand the DNA damage and repair mechanism among different tree species.A comprehensive analysis will be performed statistically by species, season, and year lateras we collect more data.

Impacts
What was accomplished under these goals? During the first year, we set up the research protocolsfor quantifying DNA/RNA, CPDs, and 6,4PPs to determine UV (A/B)- induced DNA damage. We studied 6 oak tree species for their DNA damage induced by cumulative exposure to seasonal UV radiation in Louisiana. The seasonal UV radiation data were obtained from the USDA UVB Monitoring and Research Program's field station located at Benhur Farm in Baton Rouge, LA. Oak species are some of the most ecologically and commercially important tree species in the Southern USA. Sixoak species were studied includinglive oak (Quercus virginiana),Nuttall oak (Q.nuttallii),sawtooth oak (Q.acutissima),Shumard oak (Q.shumardii),water oak (Q.nigra), andwillow oak (Q.phellos). Leaf samples were collected from the most sun-exposed branches, in the south direction of the trees during the spring (April), summer (August), and fall (November). For each species, DNA was extracted in quadruplicates. Leaf samples were first homogenized using Tissulyser LT (Qiagen), andDNA extraction was performed using the DNeasy Plant Mini Kit (Qiagen) and processedusing QIAcube (Qiagen). The extracted DNA was analyzed for quantity and quality using a UV Spectrophotometer Nanodrop 2000/2000c (Thermo Fisher). DNA damage products CPDs and 6,4-PPs were quantified using Enzyme-Linked Immunosorbent Assay (ELISA method) (Roza et al., 1988; Mori et al., 1991) with plates read by Microplate reader (ELX 800 from Biotek) at 492 nm. Preliminary results show that theDNA damage productCPD level in live oak was higher in spring leaf samples andlower in the summer and fall samples, indicating an increased level of UV resistance in leaves during a growing season.This may help explain why live oak leaves can retain on a tree longer than other oak species and forma semi-evergreen tree canopy.The other 5 oak species studied exhibited no significant seasonal changes in theCPD production, indicatingUV-resistance ability toward the cumulative exposure of UV radiation throughout the growing season.As trees grow in the natural environment, it is unavoidable to be subject to continuous and cumulative solar UV radiation exposure, as a result, they will always experience somelevelof UV-induced DNA damage as shown in this study for the 6 oak species. The data also showed that among the two major UV-induced DNA damage products, CPD production appeared to be more sensitive to the species and seasons than the production of 6,4-photoproducts (6,4-PPs). A comprehensive analysis will be performed statistically by species, season, and year lateras more data are to becollected. Meantime, we are in the process of establishing aprotocol to analyzethegeneral marker of DNA oxidative damage by measuring the premutagenic base 8-oxo-2'-deoxyguanosine (8-oxodG) at the different stages of the leaf development in the selected trees during a growing season. A new High-PerformanceLiquid Chromatograph and Mass Spectrometer (HPLC/MS) system has been acquired through leveraging other funding. The systemwill be used to analyze the8-oxo-2'-deoxyguanosine. We have been working with our collaborators including the USDA-UVB Monitoring and Research Program at Colorado and the USDA-ARS-Honey-Bee Lab to strengthen our research. We have established a link with Novegene USA inmeasuring protein and gene expression of UV-specific DNA polymerase and CPD- and 6,4PP-photolyase, which will help us better understand the DNA damage and repair mechanism among different tree species. We expect that UV-induced DNA damagemay vary with species and year due to environmental and climate variability.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yadong Qi, Vanessa Ferchaud, Wei Gao, and Gorden Heisler. 2023. Assessing UV Tolerance in Broadleaf Trees and Modeling Urban Tree Canopy Interception of Solar UV radiation. P 22-23. In the Abstract Program entitled " Monitoring, Modeling, and Assessing the Environmental Effects and Changes in Solar UV Radiation and Climate Workshop". Co-Organized by the USDA UV-B Monitoring and Research Program and the United Nations Environment Program Environmental Effects Assessment Panel, Sept 18, 2023. Colorado State University, Fort Collins, CO.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yadong Qi, Vanessa Ferchaud, and Wei Gao. 2023. Urban Tree Canopy Reduction of Solar Ultraviolet Radiation: Mechanism and Assessment (1358). P 27. Book of Abstracts, 2nd World Form on Urban Forests. Greener, Healthier, and Happier Cities for All. Washington DC, Oct. 16-20, 2023. https://www.worldforumonurbanforests.org/past-events/wfuf-washington-2023.html
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lakshmi Dasari, Yadong Qi, and Wei Gao. 2023. Modeling the Influence of Live Oak (Quercus virginiana) Tree Canopy on Air Temperature Reduction in Urban Environment During Clear-Sky Days. (3977). P166. Book of Abstracts. 2nd World Form on Urban Forests. Greener, Healthier, and Happier Cities for All. Washington DC, Oct. 16-20, 2023. https://www.worldforumonurbanforests.org/past-events/wfuf-washington-2023.html
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yadong Qi, Vanessa Ferchaud, Eman El Dakkak and Kit Chin. 2024. Quantifying Urban Forest Benefits on Reduction of Solar UV-A and UV-B Radiation. P272. In the Abstract Book of the 2024 ARD (Association of 1890 Research Directors, Inc.) Symposium. Nashville, Tennessee. April 06-09, 2024. Abstract and Oral Presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Vanessa Ferchaud, Yadong Qi, Eman El dakkak and Kit L. Chin. Localization of UV-Absorbing Compounds in Green Ash (Fraxinus pennsylvanica) and Nuttall Oak (Quercus nuttallii) Trees in the Temperate Deciduous Forest Biome. P523. In the Abstract Book of the 2024 ARD (Association of 1890 Research Directors, Inc.) Symposium. Nashville, Tennessee. April 06-09, 2024. Abstract and Poster Presentation
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yadong Qi, 2023. Assessment of Urban Tree Canopy Reduction of Solar UV Radiation and Mechanism of UV Tolerance in Broadleaf Trees, Invited presentation at the 8th China Forestry Science Conference  Urban Forestry Division, Harbin, China. July 29-30, 2023.