Source: LOUISIANA STATE UNIVERSITY submitted to NRP
IMPROVING THE QUALITY OF RICE BY IDENTIFYING TARGETS TO RESTRICT ARSENIC ACCUMULATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1011905
Grant No.
2017-67013-26165
Cumulative Award Amt.
$499,627.00
Proposal No.
2016-10070
Multistate No.
(N/A)
Project Start Date
Mar 1, 2017
Project End Date
Feb 28, 2022
Grant Year
2017
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Office of Sponsored Programs
Non Technical Summary
Arsenic accumulation in rice poses a serious threat to agriculture and human health in the U.S. and worldwide. Rice is a staple crop that feeds billions and is frequently grown on arsenic-contaminated lands, which reduces crop yields and exposes consumers to the toxic effects of arsenic through consumption of arsenic-laden rice grain. Hence, there is an urgent need to develop rice varieties that limit arsenic accumulation. The goal of this project is to identify the genetic factors that control arsenic accumulation in rice grain and arsenic tolerance of rice plants. To uncover these factors, we will (1) generate segregating populations from crosses between rice cultivars known for grain-arsenic accumulation and exclusion, (2) identify and use molecular markers to discover genetic factors linked to arsenic-related traits, and (3) test the impact of manipulating the expression of these factors on arsenic accumulation and tolerance. Our results will facilitate breeding of rice cultivars with increased yields and decreased concentration of arsenic in the grains. This will not only result in enhanced rice productivity, directly benefiting U.S. and global agriculture, but will also decrease consumption of arsenic-contaminated rice, which is increasingly recognized as a global health concern. The proposed project addresses the FY 2016 program area priority, Physiology of Agricultural Plants, in several ways, as it employs molecular and whole-plant approaches to 1) understand how plant growth affects productivity, 2) improve grain nutritional quality, and 3) understand mechanisms of plant responses to abiotic stress.
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20315301080100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1530 - Rice;

Field Of Science
1080 - Genetics;
Goals / Objectives
The goals of this project are to determine the factors in rice plants that control arsenic tolerance and arsenic accumulation. Four objectives will be undertaken to achieve these goals:1. Objective 1. Determine the arsenic accumulation and arsenic tolerance of progeny from two independent rice crosses (Years 1 and 2).Experiments for this objective will measure the grain-arsenic levels and straighthead tolerance of progeny derived from two crosses between parents deemed to be "arsenic-accumulators" or "arsenic-excluders" based on our preliminary work. We expect that segregating progeny from crosses between parents with divergent grain-arsenic accumulation will exhibit a range of phenotypes for grain-arsenic accumulation as well as tolerance to straighthead, a disorder in rice resulting from poor grain fill that has be linked to arsenic exposure.2. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3).Experiments for this objective will combine next-generation sequencing with bulked segregant analysis to map short DNA sequences to selected reference genomes to identify single nucleotide polymorphisms (SNPs) that will act as molecular markers leading to the identification of QTLs putatively associated with grain-arsenic accumulation and tolerance to straighthead disorder.3. Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3).We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder.4. Objective 4. Examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance (Years 1, 2, and 3).Experiments for this objective will use genetic over-expression and knock-down approaches for targeted genes to alter the arsenic accumulation and tolerance phenotypes of transgenic rice plants.
Project Methods
1. Objective 1. Determine the arsenic accumulation and arsenic tolerance of progeny from two independent rice crosses (Years 1 and 2).Experiments for this objective will measure the grain-arsenic levels and straighthead tolerance of progeny derived from two crosses between parents deemed to be "As-accumulators" or "As-excluders." For each cross, grain-arsenic levels will be determined via inductively coupled plasma mass spectrometry for approximately 300 F2s grown in flooded fields. These data will be used to select the 45 (15% selection) with highest grain-arsenic, and the 45 with lowest grain-arsenic. F2:3 progeny testing will be used to validate the high versus low F2 grain-arsenic selections. In addition to grain-arsenic phenotyping, straighthead selection will be conducted, and will be based on F2:3 progeny evaluated in a straighthead nursery.2. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3).Experiments for this objective will combine next-generation DNA sequencing with bulked segregant analysis ("QTL-Seq") to identify QTLs associated with grain-arsenic and straighthead tolerance. For each QTL-Seq experiment, the 10 to 15% of F2 individuals at each phenotypic extreme ("tail") will be identified. For grain-arsenic QTL-Seq, the 30 to 40 F2s with the highest F2 and F3 grain-arsenic levels and the 30 to 40 with the lowest grain-arsenic levels will be selected as the tails. For straighthead tolerance QTL-Seq experiments, 30 F2s selected based on replicated F2:3 evaluations will be used for each tail. Bulked DNA samples will be prepared for each tail by combining equal ratios of DNA extracted from frozen leaves of the F2 individuals found from progeny testing to comprise the tail. Five µg of DNA for each bulked DNA sample will be sequenced with single-end sequencing that is expected to yield a minimum of 220 million, 100 nucleotide reads per lane. A sequencing depth of 10x is planned for each sample, which will allow for pooling five barcoded samples in one lane. Each sample will be processed for quality checks, and high quality reads will be used for downstream analyses. Short reads will be mapped to selected reference genomes to identify single nucleotide polymorphisms (SNPs) that will act as molecular markers leading to the identification of putative QTLs.3. Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3).Experiments for this objective will use both validatedSimple-Sequence Repeat (SSR) markers and selected SNPs to identify optimum molecular markers residing near and in the QTL regions identified in objective 2. We will first select 5 to 10 SSRs dissecting each QTL with primers and physical locations available through the Gramene markers database. This will allow us to validate the QTLs in the larger F2 populations, which will also provide more precise estimates of the QTL effects and locations. For validated QTLs, additional markers will be developed using the candidate SNPs identified in objective 2 and, if necessary, the QTL regions can be amplified from the parents for sequencing. We will use genotype by sequencing (GBS) employed with a custom multiplexed library targeting selected SNP sites for high throughput screening as needed. Progeny testing for validation of straighthead QTLs will be conducted in year 3. As for testing associations with arsenic-related traits, heading dates will be collected on a per plant basis during the F2 and F3 progeny testing in flooded fields, allowing us to test for association between heading date and grain-arsenic each generation. Concentrations of arsenic-related elements (e.g. phosphorus and silicon) will be determined simultaneously with grain-arsenic, supporting testing for association between arsenic and these other elements in the grains. Immature and mature flag leaves will be collected from theappropriate plantsin order to support analysis of leaf-to-grain translocation for these additional arsenic-related elements. To test for association between grain-arsenic and straighthead, the population tails selected for one trait will be evaluated and compared for the other trait.4. Objective 4. Examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance (Years 1, 2, and 3).Experiments for this objective will make specific genetic changes via overexpression and knock-down or knock-out techniques to alter arsenic tolerance and/or accumulation. In light of the knowledge that the rice aquaporin family plays a major role in arsenic acquisition and likely translocation in planta, transgenic lines knocking down specific aquaporin genes have already been developed and confirmed in a reference rice cultivar (i.e. Nipponbare). We will also generate overexpression and knockdown lines for genes identified in this project as being within key QTLs. For rice transformation, embryogenic callus will be induced from mature seeds and used as target explants. Plants will be regenerated from positive transformants.Transgenic rice plants will be confirmed with genotyping and gene expression analyses.Homozygous lines will be selected for further evaluation. Using established pot and hydroponic growth systems, the arsenic tolerance and accumulation of all transgenic lines will be determined along with the wild types. Also, the arsenic concentrations of different tissues (roots, leaves, and seeds) will be quantified.

Progress 03/01/17 to 02/28/22

Outputs
Target Audience:The target audiences reached by our efforts during this award include the greater scientific community; research technical staff; graduate and undergraduate students; K-12 students; and the public. By attending major scientific conferences, we shared our experimental observations to the greater scientific community; particularly researchers who investigate genetic and physiological responses of plants to environmental stressors and poor growth conditions. Several graduate students were mentored and trained in a variety of key laboratory skills during this award. Finally, through our participation in the Super Science Saturday public science event and similar outreach activities, we presented research findings to more than several hundred children and parents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students were mentored directly on various aspects of this project.Also, one graduate student gained presentation experience by presenting his work at a scientific meeting. How have the results been disseminated to communities of interest?Results were disseminated to the scientific community through conference proceedings and publications. Participation in a major public outreach activity, Super Science Saturday, resulted in the dissemination of the project activities to hundreds of children (K-12) and their parents. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Grain- and leaf-arsenic concentrations of F2 progeny from two independent crosses were determined and extreme individuals were selected. Previously harvested seed samples from the two F2 populations (JxG, and HEDxCCC) were subsampled, dehulled, weighed, and analyzed for arsenic concentrations with ICP-MS. The seed had been produced using augmented design so that spatial effects on grain-arsenic could be corrected for as high-arsenic and low-arsenic selections were made. The extremes of these ranges are in line with the grain-arsenic concentrations of the parental lines. Approximately 45 high-arsenic and 45 low-arsenic F2 progeny were selected per each of the two populations. F2:3 progeny were grown under standard field conditions for grain-arsenic and F3 verifications. F3 seed from 500 F2 plants were planted as F2:3 plots, approximately 5 plants per plot, two replications each. Seed was harvested from individual F3 plants in each plot. Seed harvested from 5 F2:3 plants per each replication of progeny derived from the grain-As selections were threshed and readied for arsenic accumulation analysis via ICP-MS. F2:3 progeny were grown in a nursery treated with monosodium methanearsonate to induce straighthead. Two replications of plots were grown and rated for straighthead severity. Those data were corrected for field location (spatial) effects before making selections for extreme straighthead phenotypes. A total of six MSMA-treated paddies were required to hold two replications x 2 populations. After progeny testing the JxG mapping population, we identified 21 F2:3 progeny lines that were consistently high in grain-As compared with 18 that were consistently low in grain-As. While progeny from all 40 F3s selected for resistance to straighthead were again resistant in F3:4 progeny testing, due to milder severity of straighthead in year 2, only 12 progeny were confidently susceptible in both years. Phenotypic differences for both grain-As and straighthead response proved less divergent among the HEDxCCC progeny and parents, preventing the confident identification of progeny lines that were contrastingly high versus low for either trait over two generations. Conducting an additional year of progeny testing did not resolve the issue. We then turned our attention to one of the contingency populations, Jefferson X Core 310167 (JFx167). Objective 2. The genomes of 4 parental lines for this study (Grassy, Jefferson, Hunan Early Dwarf, and Chun Chu Cho) were re-sequenced. This was in anticipation of QTL-Seq experiments that were planned, but were unable to be achieved due to several delays during the award period. As a contingency, we initiated single nucleotide polymorphism (SNP) variant calling between Grassy and Jefferson based on the rice reference genome IRGSP 1.0. For the Jefferson x Core 310167 contingency population, as well as Jefferson x Grassy, DNA from phenotypically divergent progeny lines and their parents were genotyped for 1574 SNPs by sending leaf tissues to AgriPlex for commercial analysis; 342 of the 1574 SNPs (22%) proved polymorphic in the JFxGr population, and 241 (15%) were polymorphic among the JFx167. QTLs were identified based on non-random segregation of parental alleles between the high versus low grain-As progeny subsets, and between the StHD resistant vs susceptible progeny subsets. The data revealed locations for StHD QTL and a total of 5 grain-As QTLs, two identified among the JFxGr, and three among the JFx167. Objective 3. Identification of QTLs in chromosomal regions known to contain genes affecting the target trait increases confidence in the presence of those QTLs. Contrary to our hypothesis that we would find co-location among grain-As and StHD QTL, the single StHD QTL did not map near any of the grain-As QTLs. Three of the 5 grain-As QTLs were co-located with previously reported grain-As QTLs. Interestingly as well, none of the grain-As QTLs mapped near any of the genes previously reported to transport As across cell membranes (e.g, Lsi1, Lsi2, Lsi3, and Lsi6). The MSU-7.0 database annotated with known or predicted gene functions (https://phytozome-next.jgi.doe.gov/info/Osativa_v7_0) was searched to identify candidate genes within the grain-As and StHD QTL regions. Based on gene functions, candidate genes for all but one QTL (qAs2-2) were identified. OsStr9 is involved with response to oxidative stress, and might explain the StHD QTL identified on chromosome 6. One ABC transporter gene, has been shown to increase the transport of As into the vacuoles of rice stem cells, a process known as sequestration, and this sequestration was shown to reduce accumulation of As in rice grains. Four of the five grain-As QTLs we identified encompass genomic regions known to contain other ABC transporter genes. Other genes found in the grain-As QTL regions include proteins known to interact with heavy metals, such as Heavy metal ATPase 7 (OsHMA7) and cadmium tolerant 4 (OsCDT4). In flooded rice paddies, arsenic is known to enter plant cells as arsenous acid, therefore the Usually multiple acids move in and out transporter 5 (OsUMAMIT5) is also included as a candidate gene for qAs1. The QTLs and candidate genes we identified provide direction for further research to identify the genes and mechanisms rice plants use to limit accumulation of As in grains, and those used to confer resistance to StHD. Objective 4. We have characterized the rice PIP1;3 and PIP2;6 RNAi lines for arsenic tolerance and accumulation. PIP RNAi lines showed better growth compared to wild type plants. At 50 mM AsIII, the wild type plants were severely wilted whereas PIP RNAi lines had little or no effect on growth. For arsenite uptake and transport in RNAi lines, total As analysis showed a significant decrease of total As in the shoots of both PIP1;3 and PIP2;6 RNAi lines, whereas no significant changes of root As contents were observed. These results suggest that rice PIP1;3 and PIP2;6 are involved in As translocation from roots to shoots. Further, the root to shoot translocation factor for RNAi lines was much lower (almost half) than the control wild type plants. In order to study the effect of AsIII sensitivity/tolerance and AsIII transport and differential accumulation in roots, shoot and mature seeds, we grew the RNAi lines until maturity. RNAi lines showed better growth under AsIII toxicity compared to wild type control. Our results showed the RNAi lines for both PIP1;3 and 2;6 showed significant decreased in total As accumulation in roots, shoots and flag leaves but not difference in grains. Further confirmation of these results is in progress. We have also overexpressed OsPIP1;3 and OsPIP2;7. T3 generations plants were generated and seeds were bulked. Further analysis of the OE transgenic lines for AsIII tolerance and accumulation is currently ongoing. Finally, we studied the effect of nanoscale sulfur (NS) on rice seedlings and mature plants under AsIII and AsV stress. NS application caused a 40% increase in seedling biomass and a 26% increase in seed yield of mature plants compared to untreated control plants. AsIII exposure caused severe toxicity to rice; however, coexposure of plants to AsIII and NS alleviated As toxicity, and growth was significantly improved. Further, AsIII+NS-treated seedlings accumulated less As in root and shoot tissues than the AsIII-alone treatment. Mature plants treated with AsIII+NS produced more dry shoot biomass, seed number, and seed yield, and accumulated less total As compared to AsIII-alone-treated plants. A similar trend was observed in seedlings treated with AsV and NS. These results have significant environmental implications as NS application in agriculture has the potential to decrease As in the food chain and simultaneously enable crops to grow and produce higher yields on marginal and contaminated lands.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Genetic, chemical, and field management strategies for reducing accumulation of arsenic in rice grains. Presenter: Shannon Pinson (Co-PD). ASA-CSSA-SSSA annual meeting, October 22-25, 2017 at Tampa FL. Oral presentation and abstract published in the conference proceedings.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Rice Plasma Membrane Intrinsic Proteins Play Critical Role in Arsenite and Boron Transport and Tolerance in Plants. Presenter: Om Parkash Dhankher (Co-PD). ASA-CSSA-SSSA annual meeting, October 22-25, 2017 at Tampa FL. Oral presentation and abstract published in the conference proceedings.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Biotechnological Approaches for Improving Environmental Stress Tolerance and Food Safety. Presenter: Om Parkash Dhankher (Co-PD). N8 AgriFood International Conference held in Durham, UK; July 11-13, 2017. Oral presentation and abstract published in the conference proceedings.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ahmed Ali Meselhy, Gurpal Singh, and Om Parkash Dhankher. Characterization of rice plasma membrane intrinsic protein OsPIP1;3, OsPIP2;7, and their roles in arsenic and boron transport in rice plants. American Society of Plant Biologist Northeast Section (NEASPB) annual meeting, UMass Amherst, April 28-29, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pinson, S.R.M., J.D. Edwards, D.J. Heuschele, L Tarpley, C.E. Green and A.P. Smith. Genes and physiological factors associated with natural variation in rice arsenic concentrations. International Rice Research Conference 2018, Singapore, Oct. 14-17, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Pinson, S.R.M., J.D. Edwards, A.K. Jackson, D.J. Heuschele, J.Y. Barnaby, L. Tarpley, C.E. Green, E.E. Codling, and A.P. Smith. 2020. Genetic loci and agronomic traits impacting grain-arsenic concentrations revealed by GWA and biparental QTL analyses. 38th Rice Tech. Work. Group Meet., Orange Beach, AL, Feb. 24-27, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ahmed Ali, Sudhir Shrma, Om P Dhankher. Two Plasma Membrane Intrinsic Protein, OsPIP1;3, OsPIP2;6, are Involved in Arsenic and Boron Transport in Rice. Plant Biology 2019 meeting, August 7-11, 2019, San Jose CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Om Parkash Dhankher, Ahmed Ali and Sudhir Sharma. RNAi-Mediated Inactivation of Rice Plasma Membrane Intrinsic Proteins, OsPIP1;3, OsPIP2;6, Showed Decreased Accumulation and Enhanced Tolerance to Arsenic. CSSA/ASA/SSSA annual meeting, November 9-13, 2019, San Antonio TX.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ahmed G Meselhy, Sudhir Sharma, Zhi Guo, Gurpal Singh, Haiyan Yuan, Rudra D. Tripathi, Baoshan Xing, Craig Musante, Jason C. White, and Om Parkash Dhankher. Nanoscale Sulfur Improves Plant Growth and Reduces Arsenic Toxicity and Accumulation in Rice (Oryza sativa L.). Environ. Sci. Technol. 2021, 55, 13490-13503. https://doi.org/10.1021/acs.est.1c05495
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Om Parkash Dhankher and Ahmed Ali. Developing strategies for phytoremediation and limiting arsenic accumulation in rice. International Symposium on "Advances in Plant Biotechnology and Genome Editing." India, April 8-10, 2021 (Virtual Conference), Oral Keynote Talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ahmed Ali and Om Parkash Dhankher. Two Plasma Membrane Intrinsic Protein, OsPIP1;3, OsPIP2;6, are Involved in Arsenic Transport in Rice. ASA, CSSA and SSSA International Annual Meetings, Salt Lake City, Utah- November 7-11, 2021 (Poster presentation).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Heuschele, D.J., S.R.M. Pinson, and A.P. Smith. 2017. Metabolic responses to arsenite in rice seedlings that differed in grain arsenic concentration. Crop Sci. 57:2671-2687. https://doi.org/10.2135/cropsci2016.06.0493
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pinson, S.R.M., J.E. Edwards, D.J. Heuschele, L. Tarpley, C. Isbell, C.E. Green, A.P. Smith. 2018. Genetic and physiological relationships between grain arsenic and resistance to straighthead. Proc. 37th Rice Tech. Work. Group Meet., Long Beach, CA, p. 166. Feb. 19-22, 2018.


Progress 03/01/20 to 02/28/21

Outputs
Target Audience:The target audiences reached by our efforts during this reporting periodinclude the greater scientific community; research technical staff; graduate and undergraduate students. By attending virtual scientific meetings, we have shared our experimental observations to the greater scientific community; particularly researchers who investigate genetic and physiological responses of plants to environmental stressors and poor growth conditions. Several graduate students were mentored and trained in a variety of key laboratory skills during this reporting period. Changes/Problems:Because of delays resulting from government shutdowns and the COVID pandemic we requested a second no-cost extension to the award duration. What opportunities for training and professional development has the project provided?Three graduate students and 1 undergraduate student were mentored on variouis aspects of this project..One graduate student gained presentation experience by presenting his work at a scientific conference. How have the results been disseminated to communities of interest?During this reporting period, the results were disseminated to the scientific community through two conference proceedings and one published manuscript. What do you plan to do during the next reporting period to accomplish the goals?We will use QTL-sequencing to identify SNPs in the progeny ("tails") that are linked to the grain-arsenic and/or straighthead phenotypes. We have already sequenced the four parental lines and are waiting for grain-arsenic and straighthead phenotypes among the progeny to be verified.We will validate the QTLs identified from QTL-sequencing and evaluate relationships among arsenic-relatedtraits. We will continue to examine the impact of manipulating expression of PIP genes and other candidate genesin arsenic accumulation andtolerance.

Impacts
What was accomplished under these goals? Objective 1. Determine the arsenic accumulation and arsenic tolerance of progeny from two independent rice crosses (Years 1 and 2) - 95% completion.All grain data is presently undergoing variance analysis to measure, then statistically adjust the data for errors (slight increases or decreases in concentrations) caused by field location. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3) - 30% completion.Because of the delay in obtaining F2:3 grain data to confirm the "tails" to be sequenced (i.e. those progeny with the most extreme phenotypic differences related to straighthead and/or grain-arsenic), the planned QTLsequencing experiments were also delayed. Due to additional delays with the COVID pandemic, werequested a second no-cost extension for this award. In the meantime, we have re-sequenced the genomes of the 4 parental lines for this study: Grassy, Jefferson, Hunan Early Dwarf, and Chun Chu Cho and have defined single nucleotide polymorphism (SNP) variants between each pair of parental lines. Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3) - 0% completion.Because of multiple delays due to government shutdowns and the COVID pandemic,we have not yet initiated this objective, and have requested a second no-cost extension. Once the F2:3 grain data are available, we will commence with QTL-sequencing experiments in earnest. We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder. Objective 4. Examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance (Years 1, 2, and 3) - 60% completion.We have identified and characterized PIP RNAi lines for OsPIP1;3 and OsPIP2;6 in rice. We have obtained at least 2 independentT3 homozygous lines for each gene from T2 heterozygous rice seeds. The RNAi lines grew slightly better on arsenicand had denser and longer roots compared to wild type controls. Total arsenic analysis showed a significant decrease of total As in roots and shoots compared to controls. We alsogrew the RNAi lines and WT controls in small pots filled with Cornell mix rice soil amended with 20uM AsIII until maturity. The RNAi lines showed better growth under AsIII toxicity compared to wild type controls. Currently we are in the process of obtaining more transgenic lines and further analyzing all RNAi lines. In addition to the RNAi silencing lines, we have also generated transgenic PIP overexpression lines forOsPIP1;3 and OsPIP2;6, which are currently being analyzed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ahmed G Meselhy, Sudhir Sharma, Zhi Guo, Gurpal Singh, Haiyan Yuan, Rudra D. Tripathi, Baoshan Xing, Craig Musante, Jason C. White, and Om Parkash Dhankher. Nanoscale Sulfur Improves Plant Growth and Reduces Arsenic Toxicity and Accumulation in Rice (Oryza sativa L.). Environ. Sci. Technol. 2021, 55, 13490-13503. https://doi.org/10.1021/acs.est.1c05495
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Om Parkash Dhankher and Ahmed Ali. Developing strategies for phytoremediation and limiting arsenic accumulation in rice. International Symposium on "Advances in Plant Biotechnology and Genome Editing." India, April 8-10, 2021 (Virtual Conference), Oral Keynote Talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ahmed Ali and Om Parkash Dhankher. Two Plasma Membrane Intrinsic Protein, OsPIP1;3, OsPIP2;6, are Involved in Arsenic Transport in Rice. ASA, CSSA and SSSA International Annual Meetings, Salt Lake City, Utah- November 7-11, 2021 (Poster presentation).


Progress 03/01/19 to 02/29/20

Outputs
Target Audience:The target audiences reached by our efforts during this reporting period (year 3) include the greater scientific community; research technical staff; graduate and undergraduate students; K-12 students; and the public. By attending a major scientific conference in year 3, we have shared our experimental observations to the greater scientific community; particularly researchers who investigate genetic and physiological responses of plants to environmental stressors and poor growth conditions. Several graduate students were mentored and trained in a variety of key laboratory skills during this reporting period. Finally, through our participation in the Super Science Saturday public science event in Baton Rouge, LA, we presented an activity directly related to this project to more than several hundred children and parents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students were mentored directly on various aspects of this project, and in the future additional graduate, as well as undergraduate, students will be mentored on distinct parts of this project. Also, one graduate student gained presentation experience by presenting his work at a scientific meeting. How have the results been disseminated to communities of interest?During this reporting period, the results were disseminated to the scientific community through three conference proceedings (2 poster and 1 oral presentation + poster). Participation in a major public outreach activity, Super Science Saturday, resulted in the dissemination of the project activities to hundreds of children (K-12) and their parents. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will build on completed experiments to confirm the grain-arsenic levels and straighthead tolerance of progeny derived from two crosses between parents identified as "arsenic-accumulators" or "arsenic-excluders." We have already selected "tails" (groups of progeny with extreme phenotypes), and will confirm their phenotypes in the next year. Objective 2. We will use QTL-sequencing to identify SNPs in the progeny ("tails") that are linked to the grain-arsenic and/or straighthead phenotypes. We have already sequenced the four parental lines, and will obtain sequenced genomes for the other two parents in the coming year. Objective 3. We will begin to validate the QTLs identified from Objective 2 and evaluate relationships among arsenic-related traits. We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder. Objective 4. We will continue to examine the impact of manipulating expression of PIP genes in arsenic accumulation and tolerance. We will also begin searching for candidate genes linked to the QTLs identified in Objectives 2 and 3, for which to target in future transgenic experiments.

Impacts
What was accomplished under these goals? Objective 1. Determine the arsenic accumulation and arsenic tolerance of progeny from two independent rice crosses (Years 1 and 2) - 90% completion. In October 2019, we obtained by commercial contract grain element data on 4625 grain samples harvested from F2:3 plants in 2017, with which to validate the high vs low grain-As selections made in 2015 on F2 plants. In January 2020, we obtained grain element data on 425 samples of F3:4 plants harvested in 2018. All grain data is presently undergoing variance analysis to measure, then statistically adjust the data for errors (slight increases or decreases in concentrations) caused by field-location. In 2019, 460 F5 field plots were grown and harvested, seed not yet processed for elemental analysis. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3) - 20% completion. Experiments for this objective will combine next-generation sequencing with bulked segregant analysis to map short DNA sequences to selected reference genomes to identify single nucleotide polymorphisms (SNPs) that will act as molecular markers leading to the identification of QTLs putatively associated with grain-arsenic accumulation and tolerance to straighthead disorder. Because of the delay in obtaining F2:3 grain data to confirm the "tails" to be sequenced (i.e. those progeny with the most extreme phenotypic differences related to straighthead and/or grain-arsenic), the planned QTL-sequencing experiments were also delayed. Due to these delays, we requested a first no-cost extension for this award. In the meantime, we have re-sequenced the genomes of the 4 parental lines for this study: Grassy, Jefferson, Hunan Early Dwarf, and Chun Chu Cho. In addition, we have initiated single nucleotide polymorphism (SNP) variant calling between Grassy and Jefferson based on the rice reference genome IRGSP 1.0, and are now working on doing the same for Hunan Early Dwarf and Chun Chu Cho genotypes. Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3) - 0% completion Because of the delays described above, for which we have requested a first no-cost extension, we have not yet initiated this objective. Once the F2:3 grain data are available, we will commence with QTL-sequencing experiments in earnest. We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder. Objective 4. Examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance (Years 1, 2, and 3) - 60% completion. During the reporting period (Year 3), progress has been made on two aspects of objective 4. 1. Analysis of OsPIP1;3 and OsPIP2;6 RNAi lines: We have characterized rice PIP1;3 and PIP2;6 RNAi lines for arsenic tolerance and accumulation. The qRT-PCR analysis confirmed the loss of transcript for both genes in RNAi lines which showed significant reduction in mRNA transcripts corresponding to OsPIP1;3 and OsPIP2;6. During the current reporting period (year 3), we obtained T3 homozygous lines for each RNAi transgene from T2 heterozygous rice seeds. Each mature rice growth cycle takes 4-5 months and thus there is significant amount of time needed to grow rice for each generation. For both PIP1;3 and PIP2;6 RNAi, we obtained 2-3 individual homozygous T3 lines. The seeds of these homozygous lines were grown further for bulking the seeds. The loss of transcripts in T3 homozygous lines were again confirmed. We grew the T3 generation homozygous seeds on hygromycin and then seedlings of wild type and PIP1;3 and PIP2;6 RNAi lines were transferred on to MS media supplemented with 7.5 µM arsenite (AsIII) for As accumulation, transport and tolerance/sensitivity. Seedlings were grown for one week on AsIII media, photographed, the root and shoot length were measured. Root and shoot were harvested separately and oven dried for total As analysis. Total As accumulation in root and shoot tissues were analyzed using ICP-MS. Our data showed that when grown in Hoagland's solution supplemented with 25 or 50 µM AsIII, PIP RNAi lines showed better growth compared to wild type plants. At 50 µM AsIII, the wild type plants were severely wilted whereas PIP RNAi lines had little or no effect on growth. Total As analysis showed a significant decrease of total As in shoots, whereas the roots showed a corresponding increase in total As compared to controls. These results suggests that rice PIPs might be involved in As translocation from roots to shoots. We are further confirming these results both in MS media as well as in hydroponic systems. In order to study the effect of AsIII sensitivity/tolerance and AsIII transport and differential accumulation in roots, shoot and mature seeds, we grew the RNAi lines and WT controls in small pots filled with Cornell mix rice soil amended with 30 µM AsIII until seed maturity. RNAi lines showed better growth under AsIII toxicity compared to wild type control. After maturity, we harvested the mature seeds, roots and shoot biomass. We measured the total number of tillers, spikes, dry weight for shoot and mature seeds from each plant. Our initial results showed that RNAi lines had significantly higher number of tillers and spikes compared to wild type at 30 µM AsIII. Further, the RNAi lines also attained higher biomass and total seed weight than the wild type plants under AsIII toxicity. Total As accumulation analysis in mature seeds, shoot, roots and flag leaves is currently undergoing which will be reported in the next progress report. Further, we are repeating this growth assay for RNAi and WT lines with various concentrations of AsIII in mature plants. ?2. Overexpression of OsPIP1;3 and OsPIP2;6 in rice for improved AsIII tolerance and seed yield: We have previously showed that the overexpression of these rice PIPs genes (OsPIP1;3, OsPIP2;4, OsPIP2;6 and OsPIP2;7) in Arabidopsis provided strong tolerance to AsIII without any elevated levels of As accumulation in roots and shoots. From our preliminary data in Arabidopsis, it is evident that overexpression of these PIPs will be advantageous to improve rice yield under As stress without affecting As accumulation in seeds and biomass. Therefore, we are also interested in overexpressing OsPIP1;3 and OsPIP2;7 as candidate PIP genes in rice and their impact on rice growth, yield and As accumulation. We used the synthetic gene approach to synthesize the overexpression cassettes comprised of the promoter, coding, and terminator regions for both genes. We have cloned these two rice genes into a modified pCambia1300 under rice actin 1 (ACT1) promoter. Rice (Nipponbare) was transformed with constructs pCambia1300-OsPIP1;3 and pCambia1300-OsPIP1;3. T1 generations plants were generated and transferred to soil. These plants will mature in 3-4 months and then will be raised for T2 and T3 homozygous lines, which will be analyzed for AsIII tolerance and accumulation in hydroponic solution as well as in soil.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Pinson, S.R.M., J.D. Edwards, A.K. Jackson, D.J. Heuschele, J.Y. Barnaby, L. Tarpley, C.E. Green, E.E. Codling, and A.P. Smith. 2020. Genetic loci and agronomic traits impacting grain-arsenic concentrations revealed by GWA and biparental QTL analyses. 38th Rice Tech. Work. Group Meet., Orange Beach, AL, Feb. 24-27, 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Ahmed Ali, Sudhir Shrma, Om P Dhankher. Two Plasma Membrane Intrinsic Protein, OsPIP1;3, OsPIP2;6, are Involved in Arsenic and Boron Transport in Rice. Plant Biology 2019 meeting, August 7-11, 2019, San Jose CA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Om Parkash Dhankher, Ahmed Ali and Sudhir Sharma. RNAi-Mediated Inactivation of Rice Plasma Membrane Intrinsic Proteins, OsPIP1;3, OsPIP2;6, Showed Decreased Accumulation and Enhanced Tolerance to Arsenic. CSSA/ASA/SSSA annual meeting, November 9-13, 2019, San Antonio TX.


Progress 03/01/18 to 02/28/19

Outputs
Target Audience:The target audiences reached by our efforts during this reporting period (year 2) include the greater scientific community; research technical staff; graduate and undergraduate students; K-12 students; and the public. By attending a major scientific conference in year 2, we have shared our experimental observations to the greater scientific community; particularly researchers who investigate genetic and physiological responses of plants to environmental stressors and poor growth conditions. Several graduate students were mentored and trained in a variety of key laboratory skills during this reporting period. Finally, through our participation in the Super Science Saturday public science event in Baton Rouge, LA, we presented an activity directly related to this project to more than several hundred children and parents. Changes/Problems:Because of delays in obtaining grain-arsenic data, largely due to the unexpected need for a long bidding process in having the analysis completed, which was exasperated by the Dec 2018-Jan 2019 governmental furlough, we requested a first no-cost extension to the award duration. What opportunities for training and professional development has the project provided?What opportunities for training and professional development has the project provided? Three graduate students were mentored directly on various aspects of this project, and in the future additional graduate, as well as undergraduate, students will be mentored on distinct parts of this project. Also, one graduate student gained presentation experience by presenting his work at a scientific meeting. How have the results been disseminated to communities of interest?During this reporting period, the results were disseminated to the scientific community through two conference proceedings (1 poster and 1 oral presentation + poster) delivered at a national and international conference. Participation in a major public outreach activity, Super Science Saturday, resulted in the dissemination of the project activities to hundreds of children (K-12) and their parents. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will build on experiments completed in year 1 to confirm the grain-arsenic levels and straighthead tolerance of progeny derived from two crosses between parents identified as "arsenic-accumulators" or "arsenic-excluders." We have already selected "tails" (groups of progeny with extreme phenotypes), and will confirm their phenotypes in the next year. Objective 2. We will use QTL-sequencing to identify SNPs in the progeny ("tails") that are linked to the grain-arsenic and/or straighthead phenotypes. We have already sequenced two of the four parental lines, and will obtain sequenced genomes for the other two parents this year. Objective 3. We will begin to validate the QTLs identified from Objective 2 and evaluate relationships among arsenic-related traits. We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder. Objective 4. We will continue to examine the impact of manipulating expression of PIP genes in arsenic accumulation and tolerance. We will also begin searching for candidate genes linked to the QTLs identified in Objectives 2 and 3, for which to target in future transgenic experiments.

Impacts
What was accomplished under these goals? Objective 1. Determine the arsenic accumulation and arsenic tolerance of progeny from two independent rice crosses (Years 1 and 2) - 75% completion. During the reporting period (Year 2), a second replicate of seeds from two independent rice crosses (Jefferson x Grassy, and Hunan Early Dwarf x Chun Chu Cho) were processed, weighed, and sent out for commercial elemental analysis. The anticipated cost of analyzing the large number of samples required obtaining competitive bids before contracting for analysis. The bid process was further delayed due to the Federal government furlough Dec. 2018 - January 2019. Bids were obtained February 28, 2019, and are being processed by USDA purchasing agents. Grain data are anticipated by May 31, 2019. Due to this delay, we requested a first no-cost extension for this award. The verification of straighthead phenotypes using F4 progeny were completed in Year 2 (as anticipated). The F4 plots were planted and rated for straighthead severity during the summer of 2018. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3) - 10% completion. Experiments for this objective will combine next-generation sequencing with bulked segregant analysis to map short DNA sequences to selected reference genomes to identify single nucleotide polymorphisms (SNPs) that will act as molecular markers leading to the identification of QTLs putatively associated with grain-arsenic accumulation and tolerance to straighthead disorder. Because of the delay in obtaining F2:3 grain data to confirm the "tails" to be sequenced (i.e. those progeny with the most extreme phenotypic differences related to straighthead and/or grain-arsenic), the planned QTL-sequencing experiments were also delayed. Due to these delays, we requested a first no-cost extension for this award. In the meantime, we have re-sequenced the genomes of the Hunan Early Dwarf and Chun Chu Cho parental lines, and are preparing robust SNP maps for these genotypes. Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3) - 0% completion Because of the delays described above, for which we have requested a first no-cost extension, we have not yet initiated this objective. Once the F2:3 grain data are available, we will commence with QTL-sequencing experiments in earnest. We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder. Objective 4. Examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance (Years 1, 2, and 3) - 50% completion. During the reporting period (Year 2), progress has been made on two aspects of objective 4. 4.1. Analysis of OsPIP1;3 and OsPIP2;6 RNAi lines: We are characterizing the PIP RNAi lines for OsPIP1;3 and OsPIP2;6 in rice. The loss of transcripts for each gene in RNAi lines were confirmed using qRT-PCR. RNAi lines showed a significant reduction in mRNA transcripts corresponding to OsPIP1;3 and OsPIP2;6. We obtained T3 homozygous lines for each gene from T2 heterozygous rice seeds. For both PIP1;3 and PIP2;6 genes, we have now obtained 2-3 individual homozygous T3 lines. The seeds of these homozygous lines were grown further for bulking the seeds. We treated T3 homozygous plants with arsenite and measured their growth and arsenic accumulation. The RNAi lines grew slightly better and had denser and longer roots compared to wild type controls. Total arsenic analysis showed a significant decrease of total As in roots (ranging from 18-44% less) and shoots (ranging from 8-31%) compared to controls. In order to study the effect of AsIII sensitivity/tolerance and AsIII transport and differential accumulation in roots, shoot and mature seeds, we grew the RNAi lines and WT controls in small pots filled with Cornell mix rice soil amended with 20uM AsIII till seed maturity. RNAi line showed slightly better growth under AsIII toxicity compared to wild type control. After maturity, we harvested the mature seeds, roots and shoot biomass. We measured the total dry weight for root, shoot and mature seeds from each plant. Currently we are in the process of analyzing these data. Further, we are repeating this growth assay for RNAi and WT lines with various concentrations of AsIII till maturity. 4.2. Overexpression of OsPIP1;3 and OsPIP2;6 in rice for improved AsIII tolerance and seed yield: We previously showed that overexpression of rice PIPs (OsPIP1;3, OsPIP2;4, OsPIP2;6 and OsPIP2;7) in Arabidopsis provided strong tolerance to AsIII without elevated levels of arsenic accumulation in roots and shoots. This suggests that overexpression of these PIPs will be advantageous to improve rice yield under arsenic stress without affecting arsenic accumulation in seeds and biomass. Therefore, we are also interested in overexpressing OsPIP1;3 and OsPIP2;7 as candidate PIP genes in rice and their impact on rice growth, yield and As accumulation. We used the synthetic gene approach to synthesize the overexpression cassettes comprising the promoter, coding, and terminator regions for both genes. We have cloned these two rice genes into a modified pCambia1300 under rice actin 1 (ACT1) promoter and currently in the process of transforming rice with these synthetic gene constructs via Agrobactrium-mediated transformation method. T1 generation plants are expected in the next 4-5 months. Once obtained, these heterozygous seeds will be further screened to obtain the homozygous lines for further analysis in soil supplemented with arsenite.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ahmed Ali Meselhy, Gurpal Singh, and Om Parkash Dhankher. Characterization of rice plasma membrane intrinsic protein OsPIP1;3, OsPIP2;7, and their roles in arsenic and boron transport in rice plants. American Society of Plant Biologist Northeast Section (NEASPB) annual meeting, UMass Amherst, April 28-29, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pinson, S.R.M., J.D. Edwards, D.J. Heuschele, L Tarpley, C.E. Green and A.P. Smith. Genes and physiological factors associated with natural variation in rice arsenic concentrations. International Rice Research Conference 2018, Singapore, Oct. 14-17, 2018.


Progress 03/01/17 to 02/28/18

Outputs
Target Audience:The target audiences reached by our efforts during this reporting period (year 1) include the greater scientific community; research technical staff; graduate and undergraduate students; K-12 students; and the public. By publishing a paper and attending a major scientific conference in year 1, we have shared our experimental observations to the greater scientific community; particularly researchers who investigate genetic and physiological responses of plants to environmental stressors and poor growth conditions. Several graduate students were mentored and trained in a variety of key laboratory skills during this reporting period. Finally, through our participation in the Super Science Saturday public science event in Baton Rouge, LA, we presented an activity directly related to this project to more than several hundred children and parents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students were mentored directly on various aspects of this project, and in the future additional graduate, as well as undergraduate, students will be mentored on distinct parts of this project. How have the results been disseminated to communities of interest?During this reporting period,the results have been disseminated to the scientific community through the publication of a journal article and three oral presentations delivered at national and international conferences. Participation in a major public outreach activity, Super Science Saturday, resulted in the dissemination of the project activities to hundreds of children (grades K-12) and their parents. What do you plan to do during the next reporting period to accomplish the goals?As described in the project goals, we will continue to make progress on this project by pursuingexperiments in four objectives: Objective 1. We will build on experiments completed in year 1 to continue to measure the grain-arsenic levels and straighthead tolerance of progeny derived from two crosses between parents deemed to be "arsenic-accumulators" or "arsenic-excluders". We expect that segregating progeny from crosses between parents with divergent grain-arsenic accumulation will exhibit a range of phenotypes for grain-arsenic accumulation as well as tolerance to straighthead, a disorder in rice resulting from poor grain fill that has be linked to arsenic exposure. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3). Experiments for this objective will combine next-generation sequencing with bulked segregant analysis to map short DNA sequences to selected reference genomes to identify single nucleotide polymorphisms (SNPs) that will act as molecular markers leading to the identification of QTLs putatively associated with grain-arsenic accumulation and tolerance to straighthead disorder. Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3). We expect that selection among segregating progeny for QTL-flanking markers will yield progeny groups that differ significantly for the QTL trait, and for any associated traits, including grain-arsenic accumulation and tolerance to straighthead disorder. Objective 4. We will build on experiments completed in year 1 to continue to examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance. Experiments for this objective will use genetic over-expression and knock-down approaches for targeted genes to alter the arsenic accumulation and tolerance phenotypes of transgenic rice plants.

Impacts
What was accomplished under these goals? Objective 1. Determine the arsenic accumulation and arsenic tolerance of progeny from two independent rice crosses (Years 1 and 2) - 50% completion. During the reporting period (Year 1), progress has been made on three aspects of objective 1. 1. Grain- and leaf-arsenic concentrations of F2 progeny from two independent crosses were determined and extreme individuals were selected. Previously harvested seed samples from the two F2 populations (Jefferson x Grassy, and Hunan Early Dwarf x Chun Chu Cho) were subsampled, dehulled, weighed, and analyzed for arsenic concentrations with ICP-MS. The seed had been produced using augmented design so that spatial effects on grain-arsenic could be corrected for as high-arsenic and low-arsenic selections were made. Grain-arsenic concentrations ranged from 0.039 ppm to 0.469 ppm among the Jefferson x Grassy F2 progeny, and from 0.140 ppm to 0.583 ppm among the Hunan Early Dwarf x Chun Chu Cho F2 progeny. The extremes of these rangesare in line with the grain-arsenic concentrations of the parental lines. Approximately 45 high-arsenic and 45 low-arsenic F2 progeny were selected per each of the two populations so that with even a selection error rate as high as 50% we will be able to retain 20+ progeny with validated extreme phenotypes for the planned QTL-Seq stage. 2. F2:3 progeny from all F2s were grown under standard field conditions for grain-arsenic and F3 verifications. F3 seed from 500 F2 plants (approximately 250 per each of two populations) were planted as F2:3 plots, approximately 5 plants per plot, two replications each. Seed was harvested from individual F3 plants in each plot. Seed harvested from 5 F2:3 plants per each replication of progeny derived from the grain-As selections is now being threshed and readied for arsenic accumulation analysis via ICP-MS. 3. F2:3 progeny were grown in a nursery treated with monosodium methanearsonate (MSMA) to induce straighthead disease. Two replications of plots were grown and rated for straighthead severity. Those data are presently being analyzed/corrected for field location (spatial) effects before making selections for extreme straighthead phenotypes. Jefferson x Grassy progeny were later flowering and of more erect plant architecture than the Hunan Early Dwarf x Chun Chu Cho progeny. The later maturity of the Jefferson x Grassy population allowed it to exhibit slightly wider differences in straighthead resistance (plot ratings ranged from 1 to 7) than seen in the Hunan Early Dwarf x Chun Chu Cho progeny (ratings ranged from 2 to 6). A total of six MSMA-treated paddies were required to hold two replications x 2 populations. Objective 2. Identify Quantitative Trait Loci (QTLs) potentially linked to grain-arsenic accumulation and arsenic tolerance (Years 2 and 3) - 0% complete (this objective will be initiated in year 2). Objective 3. Validate Quantitative Trait Loci (QTLs) among additional progeny generations and evaluate relationships among arsenic-related traits (Years 2 and 3) - 0% complete (this objective will be initiated in year 2).? Objective 4. Examine the impact of manipulating expression of key genetic factors involved in arsenic accumulation and tolerance (Years 1, 2, and 3) - 25% completion. During the reporting period (Year 1), progress has been made on three aspects of objective 4. 1. Updates on OsPIP1;3 and OsPIP2;6 RNAi lines. We previously generated RNA interference (RNAi) lines for the aquaporin genes OsPIP1;3 and OsPIP2;6 in rice. We have confirmed the loss of transcript for both genes in RNAi lines. We grew T2 heterozygous rice seeds in soil for obtaining T3 seeds and identifying T3 homozygous lines for each gene. Seeds of each RNAi line were grown on MS media supplemented with hygromycin to select the transgenic lines and hygromycin resistant seedlings were grown in soil for raising T3 generation seeds. For both PIP genes we have now obtained 2-3 individual homozygous T3 lines. The seeds of these homozygous lines were grown further to bulk the seeds. Each rice growth cycle takes 4-5 months. Currently we are in the process of reconfirming the loss of transcripts for both genes in the T3 generation seeds and ready for analysis for growth in soil supplemented with arsenite for arsenic accumulation, transport and tolerance/sensitivity. 2. Overexpression of OsPIP1;3 and OsPIP2;6 in rice for improved tolerance and yield. We previously showed that the overexpression of these rice PIP genes (OsPIP1;3, OsPIP2;4, OsPIP2;6 and OsPIP2;7) in Arabidopsis provided strong tolerance to arsenite without any elevated levels of arsenic accumulation in roots and shoots. From these preliminary data in Arabidopsis, it is evident that overexpression of these PIPs will be advantageous to improve rice yield under arsenic stress without affecting arsenic accumulation in seeds and biomass. T herefore, we are also interested in overexpressing OsPIP1;3 and OsPIP2;7 as candidate PIP genes in rice and their impact on rice growth, yield and arsenic accumulation. We used the synthetic gene approach to synthesize the overexpression cassettes comprising the promoter, coding, and terminator regions for both genes. We have just received the synthetic genes and are currently in the process of transforming both synthetic gene constructs in to rice via Agrobacterium-mediated transformation. T1 generation plants are expected in 4-5 months. Once obtained, these heterozygous seeds will be further screened to obtain homozygous lines for further analysis in soil supplemented with arsenite. 3. Additional data on Arabidopsis PIP genes. We have screened additional PIP genes for their role in arsenic permeability, transport and tolerance in plants. The Arabidopsis genome has 13 PIP genes. In order to identify the role of AtPIPs in plants, we obtained the Arabidopsis T-DNA insertion lines for four AtPIPs (AtPIP1a, AtPIP1;2, AtPIP1;3, and AtPIP2;3). Seeds of all four T-DNA lines were grown in soil and screened for homozygosity and loss of transcripts were confirmed using RT-qPCR. The homozygous lines were grown in MS media supplemented with arsenite and growth parameters including biomass, root length and total arsenic accumulation in root and shoot tissues were analyzed. The preliminary results are very interesting. AtPIP1a knock-out plants when grown on media supplemented with 25 or 35 mM sodium arsenite, showed severe growth inhibition and attained significantly lower biomass compared to control plants, whereas, AtPIP1;2, AtPIP1;3, and AtPIP2;3 knock-out plants grown on arsenite showed strong tolerance and plants grew significantly bigger. These plants attained 2-3 fold more biomass compared to control plants. These lines were further analyzed for total arsenic accumulation in both root and shoot tissues. Preliminary results showed T-DNA lines for AtPIP1;3 and AtPIP2;3 showed significantly lower arsenic accumulation in both root and shoot tissues, although results were more pronounced for root tissues. Total arsenic accumulation analysis in AtPIP1a lines is currently in progress.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Title: Genetic, chemical, and field management strategies for reducing accumulation of arsenic in rice grains. Presenter: Shannon Pinson (Co-PD). ASA-CSSA-SSSA annual meeting, October 22-25, 2017 at Tampa FL. Oral presentation and abstract published in the conference proceedings.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Title: Rice Plasma Membrane Intrinsic Proteins Play Critical Role in Arsenite and Boron Transport and Tolerance in Plants. Presenter: Om Parkash Dhankher (Co-PD). ASA-CSSA-SSSA annual meeting, October 22-25, 2017 at Tampa FL. Oral presentation and abstract published in the conference proceedings.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Title: Biotechnological Approaches for Improving Environmental Stress Tolerance and Food Safety. Presenter: Om Parkash Dhankher (Co-PD). N8 AgriFood International Conference held in Durham, UK; July 11-13, 2017. Oral presentation and abstract published in the conference proceedings.