Source: PENNSYLVANIA STATE UNIVERSITY submitted to
IDENTIFICATION OF EPIGENETICALLY MODIFIED GENES IN MAIZE AND THEIR ROLE IN TRAIT STABILITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1010654
Grant No.
(N/A)
Project No.
PEN04613
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2016
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Project Director
Chopra, SU.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Plant Science
Non Technical Summary
This project aims at understanding genetic and environment interactions that are of epigenetics in nature. Project will study mechanisms of stable and unstable inheritance of traits that are of importance in agriculture. The project combines molecular biology aspects and uses cutting edge techniques of use of molecular markers as well as next generation sequencing methodologies to characterize whole genome transcriptome, DNA methylation and chromatin.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011510104025%
2011510108025%
2011510102025%
2011510113015%
2011510108110%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology; 1081 - Breeding; 1080 - Genetics; 1020 - Physiology;
Goals / Objectives
Regulation of gene expression in eukaryotes is controlled by molecular mechanisms which can restrict the expression of genes to a specific signal, developmental stage, tissue or cell type. Such patterns of expression can be maintained both as stable and unstable forms, and may be associated with the expression of specific alleles or epialleles. One mechanism that allows diversification of form and function is epigenetic regulation. Epigenetic marks that are of transient nature are probably widespread in the plant kingdom but are rare to isolate because of loss of the effect from one generation to the next. It is thus highly desirable to study epigenetic alleles that can respond to genetic factors/modifiers, which can heritably alter the expression pattern of these alleles. The proposed research project will build basic understanding as to what causes the mitotic progression and meiotic instability of such epigenetically regulated gene/alleles. This project is focused towards understanding the function of genetic modifiers that regulate gene expression via epigenetic pathways. Such modifiers can then be used in breeding programs for specific agronomic traits. Goals of this project are:1. Mapping and cloning of genetic and epigenetic modifiers of stress tolerance in maize.2. Characterization of genes and the epigenetic marks of genic sequences that participate in RdDm and/or non-RdDm.3. Develop epi-RILs impacted by specific epigenetic modifiers that alter RdDM or non-RdDM types of modifications in underlying target genes.4. Identification of epi-mutants in genes that contribute to drought stress tolerance and plant developmental defects5. Understanding differential DNA methylation mechanisms for whorl-chewing fall armyworm, Spodoptera frugiperda in maize.
Project Methods
Obj 1. Segregating populations have been developed in which epigenetic modifiers mop1 (RdDm) and Ufo1 (non-RdDm) along with their reporter genes have been introgressed in different maize genetic backgrounds. These F2 populations will be screened using molecular markers for the identification of affected traits. Focus will be on drought, flowering time, insect herbivore and plant growth and developmental processes. Four phenotypes in the field grown populations will used to develop genetic associations: a. photobleaching, b. flowering timing (silk emergence and pollen shedding) c. rapid growth syndrome and d. Fall armyworm feeding. Desirable introgressions will be carried further in the next generation by selfing as well as outcrossing to the recurrent parents to develop RILs and backcross populations, respectively. In addition, plant tissues from contrasting RIL introgressions carrying modifiers Ufo1 and/or mop1 either individually or together as double mutants will be subjected to molecular analysis.Obj 2. We have identified several endogenous as well as transgenic alleles/ epi-alleles that phenotypically report the presence or absence of the two modifiers that participate in either RNA directed DNA methylation (RdDM) and/or non-RdDM. We will thus first make use of these two maize epigenetic modifiers to understand the differential mechanisms that operate in RdDM and non-RdDm pathways at a single locus. Global changes in transcription (mRNA and small RNA), and DNA methylation of different will be characterized. RNA-seq and smRNA-seq will be performed on pericarp tissue to distinguish RdDM and non-RdDM pathways that are operating in different alleles of a single locus.Obj 3. To find map genes that act as locus specific genetic and/or epigenetic modifiers, epiRILs will be used. A set of epiRILs is being developed from BC1F1s impacted by Ufo1-1 and mop1-1. Maize epi-RILs impacted with these specific modifiers will help identify non-RdDM and RdDM processes, respectively. These epiRILs (F7/F8 generations) will be first used to assess the DNA methylation differences at the reporter loci/alleles. Further, suite of unique genes impacted by DNA methylation in these epiRILs will be identified in conjecture with results obtained from objective 2. These epiRILs will ultimately identify underlying loci that are impacted either by RdDM or non-RdDM types of DNA methylation modifications in the selected four traits of interest (see next approach).Obj 4. Phenotypic selections made under objective 1 will be linked with different responses in maize. Photobleaching of young leaves, flowering time, rapid growth syndrome and fall armyworm feeding will be used as phenotypic markers to further screen epiRILs. Identification of epi-mutants in genes that contribute to drought and fall armyworm stress tolerance and plant developmental defects will be further identified from epiRILs as well as the results obtained from RNAseq, sRNAseq and BSseq datasets.obj 5. A correlation between resistant epiRILs and JA levels will be developed. Gene expression and differential DNA methylation of MIR1-CP and other JA genes involved in the herbivore defense pathways mediated by small (sRNAs) will be studied (Huang et al. 2016). Marker genes in the JA pathway that will be specifically evaluated include myc, coi1, lox3, aos and opr7.These Efforts will provide new knowledge about the stability of gene expression of complex traits that are regulated via genetic, environmental and epigenetic regulatory mechanisms. These outputs will be evaluated by sharing the knowledge of this basic research during scientific meetings, and by publishing results in peer reviewed journals as research manuscripts.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:1. Corn seed company scientists were involved to discuss the project to produce additional mutants using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas)9 technology. 2. Weed scientists and professionals were involved to study the effect of herbicides and compare those with a genetic mutation that produces similar defects of buggy whip plants in the farmers' fields. 3. We interacted with university colleagues and identified epigenetic components of maize seed development. 4. We consulted a soil microbiologist to understand the role of flavonoids and sugar belowground in manifesting epigenetic changes above ground. 5. Collaborated with researchers in Donald Dansforth Plant Science Center to dissect the pathway of siRNA silencing Changes/Problems:Objective 5 was terminated in 2017 because of the retirement of Dr. Dawn Luthe in Spring 2017. What opportunities for training and professional development has the project provided?Undergraduate and graduate students received training in the areas of plant genetics and epigenetics. Students also participated in and learn field-based hybridization experiments. High throughput genotyping methods were used to perform to screen several thousands of plant populations to perform linkage mapping. Students worked with the postdoctoral fellows and learned DNA methylation and Chromatin modifications.Students presented their research results at scientific conferences. Graduate students availed opportunities to mentor undergraduates. Graduate students attended grant writing workshops and wrote competitive grants. How have the results been disseminated to communities of interest?1. Scientific conference presentations. 2. Peer-reviewed publications in scientific journals. 3. Presentation of crop biotech and epigenetic-related topics for undergraduates and graduate students. 4. Published media and news reports. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Mapping and cloning of genetic and epigenetic modifiers of stress tolerance in maize. Under thisobjective,we focussed on maize (Zea mays L.) epigeneticmodifier unstable factor for orange1 (ufo1) identified as accession GRMZM2G053177. A Ufo1-1allele is a spontaneous dominant mutation.We recently cloned ufo1, a Poaceae-specific gene expressed solely during seed development in maize. In order to validate the candidate gene,an overexpression construct using the GRMZM2G053177 coding sequence and 3' UTR from the Ufo1-1 transcript, driven by the maize ubiquitin promoter was introduced into maize. T0 plants were then crossed with different inbreds carrying P1-wr (B73, W23, and W22).Ufo1-1manifests several of its phenotypes by upregulating that maize pericarp color1 (p1), a Myb transcription factor. Using P1as a reporter, we developed an assay to screen seedlings for an active Ufo1-1 phenotype by assessing the production of flavan-4-ols, the precursor of p1 induced phlobaphenes pigments in plant body and pericarp as well as in seedlingcoleoptiles. We found that B73 and Hi-II (negative control)both produce light-dependent anthocyanin in coleoptiles while W23 and W22 do not produce any flavonoid pigments in the coleoptiles. The presence of phlobaphene precursors was only found in Ufo1-1 lines but not in silent U-S or segregating wild-type lines. This demonstrates that ectopic expression of p1 in the coleoptile is associated with the native Ufo1-1 allele. We further characterized Ufo1-1 and a loss-of-function Ds-GFPinsertion allele (ufo1-Dsg) to decipher the role of ufo1 in maize. We found that both ufo1 mutant alleles impact sugars, hormones and have defects in the basal endosperm transfer layer (BETL) and adjacent cell types. The Ufo1-1 BETL had reduced cell elongation and cell wall ingrowth, resulting in cuboidal-shaped transfer cells. In contrast, the ufo1-Dsg BETL cells showed a reduced overall size with abnormal wall ingrowth. Expression analysis identified the impact of ufo1 on several genes essential for BETL development. The overexpression of Ufo1-1 in various tissues leads to ectopic phenotypes, including abnormal cell organization and stomata subsidiary cell defects. Interestingly, pericarp and leaf transcriptomes also showed that as compared to wild type, Ufo1-1 had an ectopic expression of endosperm-development specific genes. This study shows that Ufo1-1 impacts the expression patterns of a wide range of genes involved in various developmental processes. 2. Characterization of genes and the epigenetic marks of genic sequences that participate in RdDm and/or non-RdDm. Genetic mapping and analysis of transcriptome data highlighted the importance of the maize ufo1 gene as a major modifier of genes involved in maize endosperm development. A k-mer approach was used to develop de novo assembly in order to enrich transcripts that were specifically expressed in RNA-seq libraries prepared from ZmUfo1-1 tissues as compared to wild type. 3. Develop epi-RILs impacted by specific epigenetic modifiers that alter RdDM or non-RdDM types of modifications in underlying target genes.Stable RILs have been developed and are being used for the characterization of expressed and silent epialleles in maize. A DNA methylation profile from two extreme RILs provided preliminary results on changes in CG, CHG, and CHH methylation at transposon and repeat sequences. We also developed near-isogenic lines in maize and sorghum with various levels of flavonoids and tested these lines for plant health and protection against abiotic stress and biotic stress tolerance. For the abiotic stress, we used late-season frost and for biotic stress, we used Corn Leaf Aphid infestation. Our results indicate that the presence of flavonoid compounds has a positive effect on plant health. The maize lines developed with varying profiles of flavonoids were also used for mice studies to understand the role of flavonoids in the gut health of mice while feeding them high fat and high-sugar diet. Our results show that the presence of flavonoids ameliorates the effect of a high fight diet via reducing inflammation of the intestine. The NILs, RILs, and EpiRILS developed will now be used in the next phase of this project to identify biochemical markers including plant hormones and sugars that are important for plant health. 4. Identification of epi-mutants in genes that contribute to drought stress tolerance and plant developmental defects. Maize plants carrying the Ufo1-1 allele are affected in growth and development as well as show stressed-like phenotypes. These several defects are in the form of plant stunting, leaf rolling, and plant height. Weshowed that plant bending could be due to reduced accumulation of lignin in Ufo1-1 plants. The effect of Ufo1-1 on plant height shows10 inches shorter on average than their wild-type siblings in a W23 backcross population. The reduced height was due to reduced internode length, while the number of internodes remained the same. Overall, Ufo1-1 plants have reduced aboveground and belowground biomass. Additionally, silk emergence was delayed by about 3 days in Ufo1-1 plants from the same population, indicating that there is some effect on flowering.The RNA-seq experiments were performed on various tissues to understand and identify stressed-induced pathways/genes enriched in theUfo1-1transcriptome. In the vegetative tissues (seedling and leaf), there are two broad categories of GO terms highly upregulated, response to stress and transcription. The coordinated regulation of several categories such as response to high light intensity (GO:0009644), heat (GO:0009408), hydrogen peroxide (GO:0042542), cold (GO:0009409), and hormone stimulus (GO:0032870) suggest that Ufo1-1 plants are under stress even though they were under the same conditions as the wild type controls. The similarity of up-regulation of the 'response' GO categories and the transcription GO categories suggests that up-regulation of transcription factors could be a part of the response to stress.Within the broader GO category of response to stress (GO:0006950), the seedling transcriptome had 135 genes up-regulated versus 44 downregulated, while in the leaf transcriptome there were 175 genes up-regulated versus 83 down. The genes in this category affected by Ufo1-1 include many encoding transcription factors including MYBs, heat shock proteins, peroxidases, serine/threonine protein kinases, NB-ARC disease resistance proteins, and several genes involved in carbohydrate/sugar metabolism among others.In pericarp, WRKY, bHLH, NAC, and MYB were the top families.We have further characterized the core set of differential expressed genes (DEGs) which are shared among all three tissues and found it to contain a high number of genes related to stress responses. 5. Understanding differential DNA methylation mechanisms for whorl-chewing fall armyworm,Spodoptera frugiperdain maize.We have determined that the type of defenses elicited by caterpillar frass depend on its eating habit. Frass from caterpillars that feed in enclosed structures like corn whorls or tomato fruits elicits pathogen defenses, whereas frass from caterpillars that feed on leaves tends to induce herbivore defense pathways.Network analysis of high throughput proteomic data indicates that western corn rootworm infestation leads to the elaboration of herbivore defenses in roots, while the infestation appears to activate signaling pathways in aboveground tissues. Analysis of herbivore defense response pathways in NAM founder lines indicates that insecticidal protein Mir1-CP is not synthesized by these lines and that its accumulation is unique to the Mp708 inbred.(Note: This objective was performed by Dr. Dawn Luthe and because of her retirement in Spring 2017, this objective was dropped).

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chatterjee D, Wittmeyer K, Lee TF, Cui J, Yennawar NH, Yennawar HP, Meyers BC, Chopra S. 2021. Maize unstable factor for orange1 is essential for endosperm development and carbohydrate accumulation. Plant Physiology, 186 (4):19321950
  • Type: Books Status: Published Year Published: 2021 Citation: Plant Biotechnology II Experience and Future Prospects. Editors:�Ricroch, Agnes,�Chopra, Surinder,�Kuntz, Marcel (Eds.) Springer 2021. https://www.springer.com/gp/book/9783030683443#aboutBook
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wu B, Li S, Chang H, Anderson R, Chopra S, Reddivari L. Maize Flavan-4-ols and Anthocyanins Alleviated Dextran Sulfate Sodium-Induced Colitis in Mice via Intestinal Barrier Function Restoration. Current Developments in Nutrition. 2020 Jun;4(Supplement_2):488.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Elango, D., Chopra, S., and Roth, G.W., 2020. Seeding and nitrogen fertilization effects on the yield and quality of brachytic dwarf brown midrib forage sorghum hybrids.�Crop, Forage & Turfgrass Management,�6(1), p.e20067. doi:10.1002/cft2.20067
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Elango D, Xue W, Chopra S. 2020. Genome-wide association mapping of epi-cuticular wax genes in Sorghum bicolor. Physiology and Molecular Biology of Plants. Aug;26(8):1727-37. https://link.springer.com/article/10.1007/s12298-020-00848-5
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Elango, D., Sandoya, G., and Chopra, S. 2021. Techniques and Tools of Modern Plant Breeding. In Plant Biotechnology, Experience and Future Prospects. Editors:�Ricroch, Agnes,�Chopra, Surinder,�Kuntz, Marcel (Eds.) Springer https://www.springer.com/gp/book/9783030683443
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Wu, B., Vanamala, J., Chopra, S., and Reddivari, L. 2021. Near-isogenic lines as powerful tools to evaluate the effect of individual phytochemicals on health and chronic diseases. In Plant Biotechnology II, Experience and Future Prospects. Editors:�Ricroch, Agnes,�Chopra, Surinder,�Kuntz, Marcel (Eds.) Springer.https://www.springer.com/gp/book/9783030683443
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wu B, Bhatnagar R, Indukuri VV, Chopra S, March K, Cordero N, Chopra S, Reddivari L. 2020. Intestinal mucosal barrier function restoration in mice by maize diet containing enriched flavan-4-ols. Nutrients. Apr;12(4):896. �https://doi.org/10.3390/nu12040896
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wu B, Chang H, Marini R, Chopra S, Reddivari L. Characterization of Maize Near-Isogenic Lines With Enhanced Flavonoid Expression to Be Used as Tools in Diet-Health Complexity. Frontiers in plant science. 2021 Jan 18;11:2249. 10.3389/fpls.2020.619598
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Po-Hao Wang, Kameron T. Wittmeyer, Tzuu-fen Lee, Blake C. Meyers, and Surinder Chopra 2017. Overlapping RdDM and non-RdDM mechanisms work together to maintain somatic repression of a paramutagenic epiallele of maize pericarp color1 PloS One, doi:10.1371/journal.pone.0187157.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Roach, E., Duicker, S., and Chopra S. 2016. Soil Management Affects Expression of Genes Involved in Carbon and Nitrogen Metabolism in Zea mays. Crop Science. doi:10.2135/cropsci2015.08.0488
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Cui, Jin; Wittmeyer, Kameron T.; Xue, Weiya; Tan, Qixian; Lee, Tzuu-fen; Meyers, Blake C.; Chopra, Surinder 2016. Transcriptome Analysis of Ufo1 Identify Epigenetically Impacted Genes. The 58th Maize Genetics Conference, March 17  20, Jacksonville, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Qixian Tan, Q;, Wittmeyer, K; Lee, T-f; Meyers, B; Chopra, S. 2016. Transgenerational Epigenetic Regulation in Maize: Characterization of a Silent Allele of Unstable factor for orange1. Poster presented at the Graduate and Undergraduate Gamma Sigma Delta Research Expo. March 24, 2016, Penn State University.


Progress 10/01/19 to 09/30/20

Outputs
Target Audience:1. Corn seed company scientists were involved to discuss the project to produce additional mutants using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas)9 technology. 2. Weed scientists and professionals were involved to study the effect of herbicides and compare those with a genetic mutation that produces similar defects of buggy whip plants in the farmers' fields. Changes/Problems:We are no longer working towards goal 5 as that was part of another faculty, Dr. Dawn Luthe, who retired several years ago. What opportunities for training and professional development has the project provided?1. Undergraduate and Graduate students received training in epigenetics and molecular biology. 2. Students presented their research results at scientific conferences. 3. Graduate students availed opportunities to mentor undergraduates. 4. Graduate students attended grant writing workshops and wrote competitive grants. How have the results been disseminated to communities of interest?1. Scientific conference presentations 2. Peer-reviewed publications in scientific journals 3. Presentation of crop biotech and epigenetic related topics for undergraduates and graduate students 4. Published media and news reports What do you plan to do during the next reporting period to accomplish the goals?Our results so far point out that the maize epigenetic modifier Ufo1has a specialized role andits overexpression leads to physiological stress in maize. These results also indicate thatufo1may be involved in the transcription of certain important stress-related genes. DNA methylation (Goal 5) and small RNA profiling and involvement of RNA-directed DNA methylation (RdDM) pathway through small RNA-based mechanisms seem to play a role in maize Ufo1 mutation. To further identify interacting partners of Ufo1, we started Yeast-2-Hybrid experiments, but the results are not yet available.

Impacts
What was accomplished under these goals? Goal 1: Additional epigenetics mutations were identified in maize. A maize mutation was identified for the publicly available transposon (Dsg) insertional database. Characterization of the Unstable factor for orange1(Ufo1)-Dsg mutation confirmed the seed development defect caused by the Ufo1 gene in maize. Goal 2: Analysis of transcriptome data highlighted the importance of the maize Ufo1gene as a major modifier of genes involved in endosperm development. Goal 3: Stable recombinant inbred lines(RILs) have been developed and are being used for the characterization of expressed and silent epialleles in maize. A DNA methylation profile from two extreme RILs provided preliminary results on changes in CG, CHG, and CHH methylation at transposon and repeat sequences. We also developed near-isogenic lines in maize and sorghum with various levels of flavonoids and tested these lines for plant health and protection against abiotic stress and biotic stress tolerance. For the abiotic stress, we used late-season frost and for biotic stress, we used Corn Leaf Aphid infestation. Our results indicate that the presence of flavonoid compounds have a positive effect on plant health. Goal 4: Characterization of transcriptome data showed that Abscisic acid (ABA) and drought stress genes are induced and some of these belong to dehydrins and ABA-induced genes in this stress tolerance pathway. Further, quantitative polymerase chain reaction(qPCR) validations were performed for drought tolerance genes especially the ones that have a role in sugar metabolism. Interactions between sugar and phytohormones are being characterized further.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Chatterjee, Debamalya; Wittmeyer, Kameron; Cui, Jin; Lee, Tzuu-fen; Meyers, Blake; Chopra, Surinder. 2019. Maize unstable factor for orange1 plays a role in carbohydrate accumulation and kernel development. Poster P217 presented at the 61st Annual Maize Genetics Conference. March 14  March 17, 2019 Union Station St. Louis, Missouri, USA.
  • Type: Websites Status: Published Year Published: 2019 Citation: Insect-deterring sorghum compounds may be eco-friendly pesticide. https://news.psu.edu/story/567254/2019/04/03/research/insect-deterring-sorghum-compounds-may-be-eco-friendly-pesticide
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Kariyat, R., Gaffoor, I., Sattar, S., Dixon, C., Frock, N., Moen, J., De Moraes, C., Mescher, M., Thompson, G., Chopra, S.*.�Sorghum 3-Deoxyanthocyanidin Flavonoids Confer Resistance against Corn Leaf Aphid.�Journal of Chemical Ecology, 2019; DOI:�10.1007/s10886-019-01062-8
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Mara, Cloutier, Debamalya Chatterjee, Dinakaran Elango, Jin Cui, Maryann Bruns, Surinder Chopra*. Sorghum root flavonoid chemistry, cultivar, and frost stress effects on rhizosphere bacteria and fungi. Submitted to Phytobiomes. Manuscript ID. PBIOMES-01-20-0013-R. https://doi.org/10.1094/PBIOMES-01-20-0013-FI
  • Type: Websites Status: Published Year Published: 2020 Citation: https://news.psu.edu/story/627935/2020/08/10/research/flavonoids-presence-sorghum-roots-may-lead-frost-resistant-crop Flavonoids' presence in sorghum roots may lead to frost-resistant crop


Progress 10/01/18 to 09/30/19

Outputs
Target Audience: 1. Corn and sorghum industry and scientist 2. Farmers working with heritage and specialty corn and sorghum 3. University faculty, grad students and post-doctoral fellows 4. Research associates and technical staff Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Undergraduate and Graduate students received training in epigenetics and molecular biology 2. Students presented their research results at scientific conferences 3. Graduate students availed opportunities to mentor undergraduates 4. Graduate students attended grant writing workshops and wrote competitive grants How have the results been disseminated to communities of interest?1. Scientific conference presentations 2. Outreach presentations during field days 3. Peer reviewed publications in scientific journals 4. Presentation of crop biotech and epigenetic related topics for first year undergraduates 5. Media and news reports What do you plan to do during the next reporting period to accomplish the goals?Our results so far point out that the maize epigenetic modifier ufo1 has a specialized role andits overexpression lead to physiological stress in maize. These results also indicate thatufo1 may be involved in transcription of certain important genes. To identify these interacting partners of ufo1, we plan to perform Yeast-2-Hybrid experiments along with protein pull down assays.

Impacts
What was accomplished under these goals? Goal 1: A major milestone was achieved in this project by cloning of maizeufo1gene through characterization ofUfo1-1dominant mutation. Transgenic maize plants were developed to further validate the putative candidate gene ofufo1.MaizeProUbi-Ufo1transgenic plants phenocopied the mutant phenotypes confirming the cloning ofufo1gene. Goal 2: RNA-seq, BS-seq and sRNA-seq were performed on various tissues ofUfo1-1mutant and control wild type plants. RdDm and non-RdDm related genes were identified using these high throughput sequence data sets. Goal 3: We are continuing backcrossing program to develop RILs that are epigenetically impacted by the presence/absence of Ufo1-1 dominant mutation. Goal 4: Characterization of transcriptome data showed that several abiotic stress genes are induced and some of these belong to drought stress tolerance pathway. Further, qPCR validations are in progress. DNA methylation validations were performed to confirm association of methylation profiles with expression of selected genes. To understand the function of ufo1 we have started sub-cellular localization studies. Goal 5: Was not pursued and will be discontinued as this research was performed by Dr. Dawn Luthe who retired from Penn State.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wittmeyer, K., Cui, J., Chatterjee, D., Lee, T-F., Tan, Q., Xue, W., Jiao, Y., Wang, P.H., Gaffoor, I., Ware, D. and Meyers, B.C., Chopra, S. 2018. The Dominant and Poorly Penetrant Phenotypes of Maize Unstable factor for orange1 Are Caused by DNA Methylation Changes at a Linked Transposon.�The Plant Cell,�30(12), pp.3006-3023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chopra, Surinder; Cui, Jin; Wittmeyer, Kameron; Chatterjee, Debamalya; Lee, Tzuu-fen; Meyers, Blake; Chang, Pearl; Rita Lu, Jui Hsien; Yen, Ming-Ren; Chen, Pao-Yang; Chopra, Surinder 2018. The Unstable factor for orange1 mutation alters epigenetic regulation and alternate splicing in maize genome. Talk presented at Environmental impact on epigenetic memory�session during SEB 2018, Florence, IT, 7/3/2018  7/7/2
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chopra, S. 2018. Maize and Sorghum Flavonoid Pigments: From Genetic and Epigenetic Markers to Plant Health Applications. Talk presented on 19. Dec 2018, Department�of Applied Genetics and Cell Biology? University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18 A-1190 Vienna Austria.
  • Type: Websites Status: Published Year Published: 2017 Citation: News/media report - Corn genetics research exposes mechanism behind traits becoming silent. PSU News by Jeffery Mulhollem, November 21, 2017 https://news.psu.edu/story/495374/2017/11/21/research/corn-genetics-research-exposes-mechanism-behind-traits-becoming
  • Type: Websites Status: Published Year Published: 2019 Citation: News/media report - Unraveling of 58-year-old corn gene mystery may have plant-breeding implications. Penn State News. Jeff Mulhollem January 17, 2019 https://news.psu.edu/story/554872/2019/01/17/research/unraveling-58-year-old-corn-gene-mystery-may-have-plant-breeding


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:1. Industry scientists and University Faculty 2. Undergraduate and Graduate students 3. Poster and oral presentations at scientific meetings. 4.. Submission of manuscripts for publication in peer reviewed journal. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate and undergraduate students presented their research results at National and International meetings in 2017-2018. How have the results been disseminated to communities of interest?1. Scientific conference presntations. 2. Peer reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?1. We are developing gene-gfp fusion constructs to understand the sub-cellular function of our genes of interest by studying their sub-cellular localizations. 2. Protein pull down assays will be done to isolate associated proteins for functional characterizations.

Impacts
What was accomplished under these goals? Project Impact: Here we have charcterized a classical maize mutation that shows stress symptoms in the absence of any abiotic or biotic stresses. This research has an implication in understanding the molecular basis of stress tolerance in maize and other plant species. Accomplishments: The maize mutant Unstable factor for orange1 (Ufo1) has been implicated in the epigenetic modifications of pericarp color1 (p1), which regulates the production of phlobaphenes.During this grant period we have accomplished the following: 1. We showed that the ufo1 gene maps to a genetically recalcitrant region near the centromere on chromosome 10. 2. Transcriptome analysis of the Ufo1-1 mutant allele and wild type plants identified a candidate gene using a comparative kmer based approach. 3. The candidate gene found in the mapping region is overexpressed by > 45 fold inmultiple tissues. Ectopic expression of this gene explains the dominance of Ufo11 and its phenotypes. In the mutant stock Ufo1 gene has a unique transcript which originates from within a CACTA transposon inserted in the first intron of the gene and is missing the first 4 codons of the wild type transcript. 4. Expression of ufo1 gene in the Ufo1-1 mutant is regulated by the DNA methylation status of the CACTA transposon explaining the incomplete penetrance and poor expressivity of Ufo11. 5. Transgenic overexpression lines of ufo1 phenocopy the p1 induced pigmentation in coleoptile, tassel, leaf sheath, husk, pericarp, and cob glumes. 6. In this study we have thus far uncovered the mystery of Ufo1 that has been unsolvedfor over ~50 years.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Kameron Wittmeyer, Jin Cui, Debamalya Chatterjee, Tzuu-fen Lee, Qixian Tan, Weiya Xue, Yinping Jiao, PoHao Wang, Iffa Gaffoor, Doreen Ware, Blake C. Meyers, Surinder Chopra (2018). Disruption of unstable factor for orange1, a novel protein in the Poaceae, triggers widespread phenotypic and molecular dysregulation. The Plant Cell.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Cui, Jin; Wittmeyer, Kameron; Lee, Tzuu-fen; Meyers, Blake; Chang, Pearl; Rita Lu, Jui Hsien; Yen, Ming-Ren; Chen, Pao-Yang; Chopra, Surinder. 2018. Maize Ufo1 mutant plays a role in epigenetic regulation and alternative splicing. Presented at the 59th Maize Genetics Conference, March 22  March 25, 2018, Palais du Grand Large, Saint-Malo, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wittmeyer, Kameron; Cui, Jin; Chatterjee, Debamalya; Tan, Qixian; Jio, Yinping; Lee, Tzuufen; Meyers, Blake; Ware, Doreen; Chopra, Surinder. 2018. The maize Ufo1 mutant results from ectopic over expression of an endosperm specific gene. Presented at the 59th Maize Genetics Conference, March 22  March 25, 2018, Palais du Grand Large, Saint-Malo, France.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chatterjee, D; Wittmeyer, K; Cui, J; Lee, T-F; Meyers, B; and Chopra, S. 2018. Maize Ufo1 mutation plays significant role in carbohydrate accumulation controlling plant development and stress response. Presented at the Annual Gamma Sigma Delta Research Exposition, March 27, 2018.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Collaboration with Dr. Doreen Ware's research group at Cold Spring Harbor Labs and USDA-ARS. Collaborations with geneticists, genomics labs and bioinformatics experts in the group of Dr. Blake Meyers, Donald Danforth Plant Science Center, St. Louis, MO. Collaborating with scientists from seed industry: Pioneer Hi-Bred International Inc., Dow Agro Sciences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students have been participating in this project. Students have obtained training in genetics and epigenetics to understand gene regulatory mechanisms. How have the results been disseminated to communities of interest?Research results been disseminated to communities of interest 1. Conference presentations 2. Publications in the peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? We are developing GFP gene tags to localize the expression of the Ufo1 gene in the plant cell. Identification of stress induced gene during heat and light stress. Development of global bisulphite sequencing database from the Ufo1 mutant and its comparison with wild types. Identification of genes regulated via RdDM and non-RdDM mechanisms during drought and heat stress.

Impacts
What was accomplished under these goals? Summary: This project focused on a dominant mutation that interacted with downstream genes to modify their expression. We used the maize pericarp color1, a MYB transcription factor as a reporter to follow the silencing and expressivity of the Ufo1-1. We have now narrowed Ufo1-1 to a single candidate gene whose overexpression is linked with P1;Ufo1 phenotypes. A transposon insertion into the first intron of the gene acts as a transcriptional enhancer and DNA methylation of the transposon is correlated with both expression of the candidate gene and Ufo1 phenotypes. 1. Transcriptome analysis: Ufo11 alters thee expression of thousands of genes in different tissues. In order to understand the effect of Ufo1-1 on the transcription of genes, we performed RNAseq experiments on three different tissues. Ufo1-1 has been introgressed into B73 or W23 and whole above ground seedlings, mature leaves, and 18 DAP pericarp were taken for experiments. Samples were sequenced with Illumina SBS technology to an average depth of ~42 million reads and minimum of ~28 million reads. Differential gene expression (FDR < 0.1) was accessed for each tissue independently. We found 2141 differentially expressed genes (DEGs) in seedlings, 2844 DEGs in leaves, and 3667 DEGs in pericarps. Leaves and pericarps show a fairly even split between up and down regulated genes with 1440:1404 and 1951:1716 respectively. Seedlings though had markedly more DEGs up regulated than down with 1614 up and only 527 down. These results indicate that Ufo1-1 has a broad effect on the transcriptome as might be expected for a mutation with so many varying pleiotropic defects. To validate the RNAseq studies, qRTPCR was performed on X genes with varying levels of differential expression. The majority of genes validated were in agreement between the RNAseq and qRTPCR. The DEGs of different tissues were compared to each other to identify them shared and unique genes in each. The majority of DEGs were unique to each tissue however, there were significant overlaps between the three tissues. 2. Ufo1 mapping. We completed the linkage mapping and identified recombinants to further narrow down the position if the Ufo1 gene. 3. Allelic variation at pericarp color1. We characterized alleles of p1 for their mechanism of epigenetic gene regulation. A p1 distal enhancer, 5.2 kb upstream of the transcriptional start site, has demonstrated variation in DNA methylation in different p1 alleles/epialleles. In addition, DNA methylation of sequences within the 3' end of intron 2 also plays a role in tissue-specific expression of p1 alleles. We showed here a direct evidence for small RNAs' involvement in regulating p1 that has not been demonstrated previously. The role of mediator of paramutation1 (mop1) was tested in the maintenance of somatic silencing at distinct p1 alleles: the non-paramutagenic P1wr allele and paramutagenic P1-rr' epiallele. The mop1-1 mutation gradually relieves the silenced phenotype after multiple generations of exposure? P1wr?mop1-1 plants display a loss of 24nt small RNAs and DNA methylation in the 3' end of the intron 2, a region close to a Stowaway transposon. In addition, a MULE sequence within the proximal promoter of P1-wr shows depletion of 24nt siRNAs in mop1-1 plants. Release of silencing was not correlated with small RNAs at the distal enhancer region of the P1-wr allele. We found that the somatic silencing of the paramutagenic P1-rr' allele is correlated with moderate reduction of CHH methylation and H3K9me2 in the distal enhancer of P1-rr'? mop1-1 plants. This study highlights that the epigenetic regulation of p1 alleles is controlled both via RdDM as well as non-RdDM mechanisms. 4. Assembley of Ufo1 genome using PacBio and NanoBio genmic strategies. We have developed a sequence contig of the Ufo1 genome using PacBio dataset. The PacBio data allowed us to compare the candidate region on chromosome 10 and we were able to identify the few major structural differences. Together with the results obtained from the transcriptome analyses and the linkage mapping, we have identified the candidate gene for the Ufo1. We characterized the expression of the candidate gene in different NILs and found that the expression is regulated via DNA methylation of the TE sequences 5' of the candidate gene.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Po-Hao Wang, Kameron T. Wittmeyer, Tzuu-fen Lee, Blake C. Meyers, and Surinder Chopra 2017. Overlapping RdDM and non-RdDM mechanisms work together to maintain somatic repression of a paramutagenic epiallele of maize pericarp color1 PloS One, doi:10.1371/journal.pone.0187157.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Roach, E., Duicker, S., and Chopra S. 2016. Soil Management Affects Expression of Genes Involved in Carbon and Nitrogen Metabolism in Zea mays. Crop Science. doi:10.2135/cropsci2015.08.0488.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Cui, Jin; Wittmeyer, Kameron T.; Xue, Weiya; Tan, Qixian; Lee, Tzuu-fen; Meyers, Blake C.; Chopra, Surinder 2016. Transcriptome Analysis of Ufo1 Identify Epigenetically Impacted Genes. The 58th Maize Genetics Conference, March 17  20, Jacksonville, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Qixian Tan, Q;, Wittmeyer, K; Lee, T-f; Meyers, B; Chopra, S. 2016. Transgenerational Epigenetic Regulation in Maize: Characterization of a Silent Allele of Unstable factor for orange1. Poster presented at the Graduate and Undergraduate Gamma Sigma Delta Research Expo. March 24, 2016, Penn State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Wittmeyer, Kameron; Tan, Qixian; Xue, Weiya; Lee, Tzuu-fen; Meyers, Blake C.; Chopra, Surinder. 2016. The mysterious Ufo1: What we have learned from global analyses. The 58th Maize Genetics Conference, March 17  20, Jacksonville, FL, USA.