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INVESTIGATOR: Milner, M.; Assmann, S. M.

PERFORMING INSTITUTION:
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK, PENNSYLVANIA 16802

EXTRA-LARGE G PROTEINS (XLGS) AND THEIR ROLE IN NUTRIENT AND HORMONE REGULATION OF LATERAL ROOT GROWTH

NON-TECHNICAL SUMMARY: To keep America a leader in agriculture, a greater understanding of basic plant processes is needed to help reduce the costly inputs farmers apply to their crops and help feed the ever increasing human population. One of the most costly inputs for farmers today is nitrogen (N) fertilizer. Globally, farmers are adding more than 100 million tons of N fertilizer to their fields every year. If farmers could reduce inputs added to their fields they could reduce costs and still maintain current crop yields. Just how a plant senses the levels of N in the soil and how perceived N levels change root architecture in response to limiting N conditions remains unclear. A better understanding of the signaling mechanism involved in N perception and uptake could result in a substantial savings for farmers. An understanding of the signaling cascade once N is perceived at the root could lead to fewer inputs and more efficient uptake of nutrients from the soil. For the current the research will focus on how a class of proteins called the XLG proteins influence N uptake from the soil as well as if all forms of N uptake are regulated by XLGs or only uptake of nitrate. We will also study how the XLGs influence the root structure to help N uptake. This will help us to understand how N is being absorbed by the root and help to engineer more efficient crops to maximize crop yield and limit excess N being added to the environment.

OBJECTIVES: Overall goal: Reduce costly N fertilizer inputs while maintaining crop yield. Supporting objectives: 1) Uncover which XLG protein(s) alter nitrogen uptake and sensing by measuring uptake and lateral root growth for different N sources and xlg mutant combinations. 2) Use RNAi to identify whether XLG regulation of nitrate response and lateral root development carries over to tomato, and whether XLG knockdown can reduce N fertilizer inputs in the field. 3) Probe alterations in auxin transport in the xlg mutants through auxin reporter gene analysis and crosses with auxin-related mutants. 4) Perform protein-protein interaction tests to assess possible XLG regulation of nitrogen- or auxin-related transcription factors.

APPROACH: Objective 1: Is altered nitrogen uptake or sensing playing a role in the increased lateral root growth in the xlg triple mutant, and is one XLG protein specifically involved in this phenotype We will test all possible combinations of xlg mutants on varying nitrate and ammonium levels to identify which gene(s) is/are involved in the larger growth and increased lateral root phenotype. Objective 2: Does lack of XLG expression in other plant species regulate lateral root branching and N fertilizer requirements We will attempt to knock out or down the xlg proteins in tomato to understand if the xlg family plays the same role in other plant species as it does in Arabidopsis. We will then test these mutants in the field to determine if we can use lower levels of N fertilizer to maintain current yields. Objective 3: Is the increased growth and lateral branching affected by altered auxin transport or auxin perception in the triple mutant We will look at the effect of knocking out the XLGs specifically involved in the increased lateral root phenotype to understand how the lack of XLG expression changes the auxin pathway in plants using microarrays to identify candidate genes that may interact with the XLG family. Objective 4: Do the XLG proteins physically interact with transcription factors regulated by N or auxin We will test known regulators of N acquisition and auxin regulation for interactions with the XLG proteins to understand at the molecular level how XLGs regulate N uptake and auxin signalling in roots.

PROGRESS: 2011/09 TO 2014/08
Target Audience: Research scientists, policy makers, lay public Changes/Problems: To generate the transgenic tomatoes took longer than expected as the genes we were trying to alter effect the plants ability to go through tissue culture. Experiments are now just starting on the material which was generated from this funding. This delayed what we could do in the time frame for which the fund was issued. We hope to publish results from the tomato aspect of the in the coming months. Protein protein interactions alone were not enough to predict nutrient pathways so we diverted some of the funds to also measure changes in gene expression to help build a better picture of how plants percieve nutrient in their enviroment. What opportunities for training and professional development has the project provided? Attended a meeting to help communicate science to the lay public and help the lay public understand why science is inportant to fund. Attended a NIFA fellows meeting in 2012 and met many other young scientist as well as program directors. This allowed the communcating of scientific ideas and exchanging of novel concepts to provide impactful science. How have the results been disseminated to communities of interest? Two publications will be submitted in the near future as Dr. Assmann and I put the final preperations on the manuscripts from data collected in the last 3 years. What do you plan to do during the next reporting period to accomplish the goals? Finish submitting the manucripts.

IMPACT: 2011/09 TO 2014/08
What was accomplished under these goals? We have made progress in understanding how plants regulate their nutrient uptake and started to identify pathways involved in sensing excess nutrients as well as defiency responses in a plants environment. This includes finding novel proteins interacting with G proteins to regulate genes involved when hormone levels are altered in plants. From this two publications will be published which will help the plant biology community to further explore how to alter nutrient sensing in plants.

PUBLICATIONS (not previously reported): 2011/09 TO 2014/08
1. Type: Journal Articles Status: Other Year Published: 2015 Citation: Milner MJ and Assmann SM. global transcription of G protein mutants reveals novel regulation of nutrition in plants. In prep.
2. Type: Journal Articles Status: Other Year Published: 2015 Citation: Ferrero A, Milner MJ, Assmann SM. Regulation of growth and fruit set in tomato by XLG proteins.

PROGRESS: 2011/09/01 TO 2012/08/31
OUTPUTS: Poster presentation at the first annual NIFA fellows meeting on Aug 16th and 17th, 2012. PARTICIPANTS: The PI Matthew Milner is a post-doctoral researcher who has learned a number of new techniques in the field of molecular biology and proteomics. He has also presented his findings at the first annual NIFA fellow meeting helping him network with other leading scientist. Dr. Sally Assmann has mentored Matt for the past year in her lab and provided guidance and oversight for the research. Boyce Thompson Institute has started to generate the RNAi lines to knock down the expression of the tomato XLGs. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2011/09/01 TO 2012/08/31
Modern agriculture requires the application of many inputs to help achieve the maximal yield. Recent research into the roles that a subclass of G proteins called Extra Large G proteins (XLG) play in plants has revealed an involvement in numerous processes including root architecture, flowering, disease resistance, and growth. In particular, when all three XLG proteins are knocked out in Arabidopsis, the plants show altered root length relative to wild type Arabidopsis under a wide range of nitrate levels, and altered transport of auxin, a hormonal regulator of root development. The overall goal of the research supported by grant 2011-67012-30722 is to reduce costly nitrogen fertilizer inputs while maintaining crop yield by gaining a greater understanding of how XLG proteins perceive or communicate the nitrogen levels to the plant and how this impacts root development and ultimately yield of the plant. We have determined that the shorter root phenotype seen in the XLG triple mutant under high nitrogen stress is related to only one of the three XLG genes in Arabidopsis. This XLG, involved in the root development sensitivity to high nitrogen, was used as the bait in a yeast two hybrid screen to identify novel interacting proteins with this XLG that may play a role in the root development phenotype seen under nitrogen stress. These novel XLG interactors are currently being characterized for their role in the shorter root growth phenotype seen in the mutant under high nitrogen stress. We also have determined that the shorter root phenotype exhibited by both the XLG triple mutant and a particular XLG single mutant under nitrogen stress is not specific to high nitrogen alone but that the root growth of both the triple XLG mutant and the single XLG mutant were also inhibited to a greater extent than in wild type plants by other macronutrients such high phosphate and high potassium. We have started to understand how the XLGs fit into the classical hetrotrimeric G protein signaling cascade to help understand how signals are communicated through the cell. A greater understanding of the intermediate proteins involved in the hetrotrimeric G protein signaling cascade may help to identify future breeding targets for the multitude of agronomically important traits related to the hetrotrimeric G proteins including salt stress, biomass production, flowering time and disease resistance. We have started to generate RNAi lines to knock down the expression of each of the XLGs in the commercial variety of tomato, NCERB1. In an attempt to find the ortholog in tomato of the Arabidopsis XLG responsible for the root sensitivity to high nitrogen these newly generated RNAi tomato lines will be assessed for their sensitivity to high nitrogen in the lab. The transgenic tomato lines will also be tested under field conditions to test if the knocked down expression of a particular XLG can alter growth characteristics, such as root growth, biomass production and yield in the field under both low and high nitrogen supplementation.

PUBLICATIONS: 2011/09/01 TO 2012/08/31
No publications reported this period