Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The undergraduate and graduate students who have worked on the project. Local scientists, who attended seminars and poster presentations from the students at Virginia Tech. Fellow researchers who are working in the same field of study or who are reading our publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students working on the AAP1 project were supervised by the graduate student Brett Shelley and were trained on molecular biology and plant phenotyping. The progress of the work of the students was monitored by weekly meetings with the PI. The students were also trained to present their results orally at weekly lab meetings, and at local conferences. How have the results been disseminated to communities of interest?Seminar and poster presentations have been delivered by students to local scientists and students during Virginia Tech organized events (VTLSS seminars, SPES seminar series, TPS Symposium, and Summer Undergraduate Symposium). Due to the COVID pandemic, no member of the Pilot lab has attended national or international conferences. What do you plan to do during the next reporting period to accomplish the goals?Goal1: We will write a manuscript to report our finding concerning the role of AAP1 in amino acid susceptibility, which will require a few more experiments with biological material already in hands. In addition, we will test the hypothesis that two amino acids from the AAP1 protein are phosphorylated and important for controlling AAP1 activity. Goal2: We will determine the protein content in the seeds of the transgenic soybean lines that are currently growing in the greenhouse, in addition to thoroughly studying the localization of the expression of the amino acid transporter GmGAPB1. During the summer, we plan to cross these lines with low and high protein accessions, get field data concerning plant vigor and seed protein content, and get homozygous lines.
Impacts
What was accomplished under these goals?
OUTCOMES. Project (1) - The aap1 mutants that bear point mutations in the AAP1 gene behave differently than the wild type and the knockout mutant. These mutants are thus not loss-of-function mutants; yet, similar to the knockout mutant, they are tolerant to high concentrations of amino acids. Two of the four corresponding AAP1 proteins also displayed altered transport properties, while the other two mutations did not affect transport. This year, we created and studied the phenotype of lines that express AAP1 in specific tissues of the root. We found that, contrary to the accepted model, the expression of AAP1 in the tissues that are supposed to mediate amino acid uptake does not render the plants susceptible to amino acid toxicity. On the contrary, expression in phloem and root tip (not participating in amino acid uptake) led to strong amino acid susceptibility. We also have confirmed most of the results that we had obtained in the previous years about the phenotype of the various mutants. Project (2) - We previously found that the GmGABP1 gene from soybean carries a sequence variation that seems to correlate with seed proteins, suggesting that this gene influences the quantity of protein stored during seed development. To test this hypothesis, we are growing transgenic soybean plants that express the two variants of the amino acid transporter GmGAPB1, and will test whether and how its altered expression affects protein content in seeds. Our preliminary data suggest that GmGABP1 is expressed in vascular tissues. IMPACTS. Our results support the hypothesis that AAP1 is mainly involved in the regulation of amino acid (and possibly nitrogen) metabolism. We conclude that AAP1 is likely a sensor and a transporter. Our work will enable us to identify genes involved in the sensing of amino acids and provide clues about the way the activity of the corresponding proteins is regulated. These genes could be used as tools to modify the allocation of amino acids in the various parts of the plants like seeds and storage organs.
Publications
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The undergraduate and graduate students who have worked on the project. Local scientists, who attended seminar and poster presentations from the students at Virginia Tech and at the University of Maryland. Fellow researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A new undergraduate student working on the AAP1 project since June 2019 was supervised by the graduate student Brett Shelley, and was trained on molecular biology and plant phenotyping. The progress of the work of the students was monitored by weekly meetings with the PI. The students were also trained to present their results orally at weekly lab meetings, and at local and regional conferences. How have the results been disseminated to communities of interest?Seminar and poster presentations have been delivered by students to local scientists and students during Virginia Tech organized events (PPWS seminar series and Summer Undergraduate Symposium). What do you plan to do during the next reporting period to accomplish the goals?Goal1: We will compare the activity of the metabolism of all the available aap1 mutants, focusing on sugar and amino acid metabolism. We also will test the hypothesis that other AAP proteins are amino acid sensors by studying the complementation efficiency of knockout mutants by the wild type and variant proteins. Goal2: We will determine the protein content in the seeds of the transgenic soybean lines, in addition to more thorough study of the line behaviors both in the greenhouse and in the field. We will also identify the tissues in which GmGABP1 is expressed.
Impacts
What was accomplished under these goals?
OUTCOMES. The aap1 mutants that bear point mutations in the AAP1 gene behave differently than the wild type and the knockout mutant. These mutants are thus not loss-of-function mutants, yet, similar to the knock-out mutant, they are tolerant to high concentrations of amino acids. Two of the four corresponding AAP1 proteins also displayed altered transport properties, while the other two mutations did not affect transport. We have shown that AAP1 needs to be expressed in the root tip to be functional, in disagreement with the current assumption that expression in the external tissues of the main root is critial for its function. We have determined the amino acid composition from 660 leaf samples taken from 165 soybean lines to seek any correlation between leaf free amino acids and seed protein content. The data are being analyzed. We have obtained from the Wisconsin Crop Innovation Center (collaboration from Dr. Jean-Michel Ane, U of Wisconsin, Madison) transgenic soybean lines that express either variants of GmGABP1 in the Williams background . IMPACTS. Our results support the hypothesis that AAP1 is not only involved in amino acid transport by the root, but also in the regulation of amino acid (and possibly nitrogen) metabolism. Our results will enable to identify genes involved in the sensing of amino acids, and provide clues about the way the activity of the corresponding proteins is regulated. These genes could be used as tools to modify the allocation of amino acids in the various parts of the plants like seeds and storage organs.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Exploring how plant nutrient transport affects resistance and susceptibility to oomycete pathogens. Sonnawala, U.; Herlihy, J.; Long, T.; Pilot, G.; McDowell, J. M. 18th Congress of International-Society-for-Molecular-Plant-Microbe-Interactions (IS-MPMI), Glasgow, SCOTLAND, 14 Jul 2019 - 18 Jul 2019. MOLECULAR PLANT-MICROBE INTERACTIONS. 32: 137-137. 01 Oct 2019
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
POSTER: Genetic evidence suggests that Arabidopsis Amino Acid Permease1 plays a regulatory role in amino acid homeostasis, potentially acting as a transceptor. Shelley, Brett; Pratelli, Rejane; Harris, Fiona; Boyd, Shelton; Pilot, Guillaume. International Workshop on Plant Membrane Biology, Scottland, UK. 08 Jul 2019
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
POSTER: Old Dog, New Tricks: Amino Acid Permease I Emerges as an Amino Acid Sensor and a Regulator of Nitrogen Metabolism. Shelley, Brett; Harris, Fiona; Boyd, Shelton; Pratelli, Rejane; Pilot, Guillaume. American Society of Plant Biologists Conference - Mid Atlantic Section, U. of Maryland, MD. 30 May 2019
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Progress 07/01/18 to 09/30/18
Outputs
Target Audience:The undergraduate and graduate students who have worked on the project. Local scientists and researchers, who attended seminar and poster presentations from the students at Virginia Tech. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The undergraduate and graduate students who worked on the project from July 2018, Fiona Harris and Brett Shelley, were trained on molecular biology and yeast techniques. The student Aparan Cheran was trained in amino acid extraction and analytical methods. The progress of the work of the students was monitored by weekly meetings with the PI. The student were also trained to present their results oraly at weekly lab meetings. How have the results been disseminated to communities of interest?Seminar and poster presentations have been delivered by students to local scientists and students during Virginia Tech-organized events (PPWS seminar series and Summer Undergraduate Symposium). What do you plan to do during the next reporting period to accomplish the goals?Goal1: We will compare the activity of the metabolim of all the available aap1 mutants, focusing on sugar and amino acid metabolism. We also will test the hypothesis that other AAP proteins are amino acid sensors by studying the complementation efficiency of knockout mutants by the wild type and variant proteins. Goal2: We will analyze the amino acid content of the soynbean leaves and perform whole genome association mapping. We also expect to get transgenic soybean plants that are being created at the University of Wisconsin, that express variants of tne GmGABP1 gene, and study these lines.
Impacts
What was accomplished under these goals?
OUTCOMES. We have confirmed in our preliminary results that the aap1 mutants that bear point mutations in the AAP1 gene behave differently than the wild type and the knockout mutant. These mutants are thus not loos-of-function mutants, yet, similar to the knock-out mutant, they are tolerant to high concentrations of amino acids. Two of the four corresponding AAP1 proteins also displayed altered transport properties, while the other two mutations did not affect transport. We have extracted amino acids from 660 leaf samples taken from 165 soybean lines to seek any correlation between leaf free amino acids and seed protein content. IMPACTS. Our results support the hypothesis that AAP1 is not only involved in amino acid transport by the root, but also in the regulation of amino acid (and possibly nitrogen) metabolism. Our results will enable us to identify genes involved in the sensing of amino acids, and provide clues about the way the activity of the corresponding proteins is regulated. These genes could be used as tools to modify the allocation of amino acids in the various parts of the plants like seeds and storage organs.
Publications
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