This project aims to contribute to the search for nonconventional crops suitable for use in regions facing diminishing quality of water and land resources. With urban expansion taking increasing amounts of prime agricultural land and freshwater resources, there is a growing need for crop plants that can be grown in areas of high salinity. This would allow agricultural crop production near oceans and in other areas that have become more saline over time due to heavy past irrigation. However, the potential of halophytes - natural flora of saline habitats - has been under-examined, and their utilization may allow production of considerable plant biomass under these conditions. This could provide great economic value to farmers in traditionally poor regions of Pakistan and other countries, but the challenge is to determine how to use them commercially and to find an appropriate niche where they could be grown efficiently to produce good quality food, fodder, medicines, oilseeds, and so forth. The researchers involved in this project will analyze three plant species with potential for use as such nontraditional crops. Beyond helping to improve the capacity of the Pakistani researchers involved and building their laboratory infrastructure, the project should also contribute to the reclamation of arid land in Pakistan for productive use by subsistence farmers and lead to new applications that can be extended to other countries as well.
At the Institute for Sustainable Halophyte Utilization in Karachi, scientists have been studying the mechanism of salt tolerance in the three species. Morphological, eco-physiological and biochemical responses of all three species when grown under no salt, optimal salt, and excessive salt conditions, including germination, growth rates and total biomass produced, photosynthesis, oxidative stress, and other properties were studied. Several manuscripts have been published over the past 3 years of this project reporting various aspects of the physiology of these and a few other halophyte species. Substantial additional information has been obtained on the mechanism of salt tolerance of these species, which is now being compiled and correlated with the gene sequence and proteome data, and will be submitted for publication soon.
In order to identify genes and proteins that are expressed under optimal salt conditions, the researchers have isolated RNA from Suaeda grown under each of the three conditions mentioned above, and the RNA was converted by reverse transcription to cDNA for Illumina sequencing. This technique (called transcriptome analysis) allows for identification of the genes that are expressed at higher levels (or lower levels) when the plants are grown under optimal salt conditions as compared to either no salt or salt stress conditions. They have completed the DNA sequencing of the Suaeda samples and are nearing completion of the computational analysis, which includes identification of the genes by comparison with genes from other species that have been sequenced. The team found a number of potentially novel genes that will be the focus of detailed analysis over the next few months. This has great potential for identifying new genes and mechanisms by which halophytes tolerate levels of salt that are toxic to most crop plants.
The researchers are currently completing the sequence analysis and have begun preparation of one manuscript to report the optimal methods for the computational analysis of the large amount of data for a new species that has not yet been sequenced. They are also preparing another manuscript to report the transcriptome of Suaeda fruticosa. They are completing the proteome analysis of plant total protein samples, which will be the subject of a third paper on the identification of genes that show substantial differential expression when the plant is grown under optimal salt conditions as compared to no salt.
During the past year one Ph.D. student and three undergraduate students have been directly trained in the U.S. lab and have contributed to this project. In addition, eight more undergraduate students working in the lab on different projects have gained an understanding of the halophyte research and its importance. The Pakistani group includes six Ph.D. students who are all working on various aspects of the project. They have held joint discussion groups of basic molecular biology principles and techniques between the two groups.
Suaeda fruticosa, Desmostachya bipinnata, and Salvadora persica.
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2011 Show summary || Hide summary || Download full report
A workshop on advances in the ecophysiology of salt tolerance was organized at the Institute of Sustainable Halophyte Utilization in Karachi April 11-13, 2011, with speakers from Pakistan, Japan, and Germany participating. Dr. Nielsen and Dr. Gerry Edwards of Washington State University presented lectures through videoconferencing, and about 50 students and faculty attended. Meanwhile, germination, growth optimization, and salt tolerance experiments on Salvadora persica continued to progress well in the Karachi lab, with almost all work on that species being completed by the end of September 2011. Co-PI Dr. Bilquees Gul began a one-year training visit to BYU at the start of June, bringing with her samples of seeds from all three species being studied. Her input was very helpful in optimizing growth conditions for the plants in the BYU greenhouse, and preliminary growth and salinity tolerance experiments are now proceeding, with the primary focus initially being Desmostachya bipinnata and samples of the other two species beginning to be harvested in late July. Dr. M. Ajmal Khan also joined the team at BYU for a one-month visit beginning in mid-August, and in September Dr. Gul took part in a week-long proteomics training course at the National Institutes of Health. In addition to the experimental work being carried out on both sides, Dr. Nielsen has delivered weekly lectures by videoconference to staff and students in the Karachi lab, with the topics for each session drawn from chapters in the classic textbook Molecular Biology of the Gene (Watson et al.). One or two more Pakistani scientists are expected to come to BYU for training in 2012 and 2013. In the coming months these researchers will be continuing their experiments on the effects of various salinity levels on plant growth, including work on optimizing protein extraction and gel electrophoresis methods. Meanwhile, they report that they have been contacted by representatives of several private companies in the United States and Pakistan interested in developing halophyte plants for commercial production.
2012 Show summary || Hide summary || Download full report
At the end of May 2012, Pakistani co-PI Dr. Bilquees Gul completed her one-year visit to BYU, during which she focused on research on the proteomics of proteins from various halophyte plants. She has prepared cDNA libraries for each of the three species included in this study (Salvadora persica, Desmostachya bipinnata, and Suaeda fruticosa) at different salt treatment levels. Illumina sequencing has been initiated on several samples to obtain transcriptome data. She and Dr. Nielsen have been collaborating with Dr. John Prince, a biochemistry professor at BYU who is assisting with the mass spectrometry and proteomics work. Although Dr. Gul has returned to Pakistan, the three researchers continue to collaborate to analyze their data, and they expect to submit a publication on the Suaeda fruticosa proteome by the end of the summer, with additional manuscripts on proteome data from the other species to follow in the coming year. Dr. Nielsen and his graduate student, Joanne Arce, will continue their sequencing and analytical efforts, while Dr. Khan and his colleagues at the University of Karachi are pursuing their experiments and gathering data on specimens of the three plant species. For the Pakistani team, their primary challenge is to gain experience with some of the novel DNA and protein sequencing approaches being used in the project as they expand their expertise from data acquisition to data analysis. As part of this capacity building effort, one or more Karachi-based faculty members and/or students are expected to visit BYU in the final year of the project.
2013 Show summary || Hide summary || Download full report
The U.S. team is using next generation RNA sequencing to analyze the expressed genes of Suaeda fruticosa a succulent halophyte (salt tolerant) species with potential as a crop for seed oil or other applications. RNA from these plants grown under three conditions of no salt (control), optimal salt, and high salt (salt shock) was purified, converted to DNA and sequenced. This data is being analyzed to identify genes that are induced or repressed in plants grown in each salt condition. One graduate student and 5 undergraduate students have been mentored as part of this project in the U.S. Dr. Nielsen continues to collaborate with Dr. John Prince, a biochemistry professor at BYU who is assisting with the mass spectrometry and proteomics work. Total protein analyses have been conducted on all three samples using the mass spectrometer and proteomics analysis identifying proteins potentially involved in salt tolerance. Initially, the focus is on Suaeda fruticosa but subsequently it will expand to the other species as well. A new graduate student, Joann Arce, has joined Dr. Nielsen’s lab at BYU and began work on the project. The research team aims to submit a manuscript on their work for publication by summer 2013 when the Pakistani PI or co-PI visit in July or August.
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