2013 Annual Report
1a.Objectives (from AD-416):
1. Generate, in collaboration with DOE-JGI, gene expression data (RNA-Seq) for approximately 180 tissues/conditions (30 in year 1, increased to 100 in year 2, and to 180 in year 3); 2. Identify and characterize tissue-specific promoters. Using the gene expression data from Activity 17 above, we will identify up to 20 different promoters in year 1 that mediate tissue or condition-specific gene expression and clone at least 10 such promoters into GUS/GFP expression vectors. These will then be tested transiently in soybean. At the end of year 2 we will continue to identify additional promoters from the gene expression atlas, clone these and test them by transient expression. By the end of year 3 we will reach our goal of screening at least 50 promoter constructs transiently.
1b.Approach (from AD-416):
The initial pilot phase of the project will target 30 conditions. These are yet to be fully defined but will include all of the major tissues of soybean (e.g., leaf, root, seed, stem, apical and root meristem, and nodules). In some cases, these tissues will be analyzed over a set time period. Additional conditions may target various biotic and abiotic stress conditions, etc. In order to maximize the uniformity of these initial samples, all plants will be grown in the Stacey laboratory (using growth chambers) and the mRNA will be isolated by a single lab member before shipping to DOE-JGI for sequencing. We plan to analyze soybean under a wide range of conditions that include various stresses (e.g., drought, pathogen infection, cold, heat, etc.), as well as various hormonal treatments (e.g., auxin, ethylene, cytokiniin, etc.). We plan to utilize the gene expression data, derived from the transcriptional gene index described above, to identify and clone promoter elements that will control gene expression in a tissue-specific or condition-specific way. For example, analysis of the currently available soybean transcriptional index (e.g., http://digbio.missouri.edu/soybean_atlas) led to the identification of roughly 9 genes that show an extremely high level (>1000-fold) of tissue specific expression. The promoters of these genes are, therefore, natural targets for cloning and use to drive tissue specific expression. A natural extension of generating the transcriptional atlas is also the ability to identify and isolate promoters that will be useful for both basic and applied studies in soybean. These promoters will provide the ability to specifically target the expression of transgenes, while also providing tools (e.g., promoter-green fluorescent protein constructs) to analyze the cellular basis of the soybean response to a variety of treatments. We will work closely to make sure that the data are directly integrated into the soybase.org site.
We have joined the Department of Energy-Joint Genome Institute (DOE-JGI) transcriptome atlas project which will measure gene expression of ten key model species in the same experimental and time course experiments. Inclusion of the model legumes soybean, medicago and bean will allow cross-species comparisons and will improve our understanding of comparative gene expression patterns, as well as gene function. Collaborating labs that are part of the DOE-JGI project agreed to perform one joint experiment to demonstrate the utility of the cross-species comparison. This experiment involves a comparison of growth under three nitrogen sources, nitrate, urea and ammonia. In addition, given that soybean develops a nitrogen fixing symbiosis with soil bacteria, we also isolated plant material from nodulated plants. All of the soybean materials for this experiment were isolated and the RNA shipped to DOE-JGI for library construction and sequencing. These RNA also provided the material to conduct a quality control study on the techniques that the larger group had agreed upon. DOE-JGI informed us that all samples passed their QC steps and the sequencing has begun.
The details of this experiment are as follows: Soybean (William 82) seeds were surface sterilized and grown for four weeks in four nutrient conditions: (a) KNO3 (b) (NH4)2SO4 (c) Control -Urea and (d) symbiotic condition without nitrate source and inoculated with B. japonicum. Three biological replicates from each tissue types (sink leaves, roots and nodules from symbiotic condition) were harvested. Total RNA and purified small RNA were extracted from the tissues (over 99 samples, including the ones mentioned below) using an agreed upon procedure. After extraction of these RNA, the samples were shipped to DOE-JGI for deep-coverage RNA-seq, small RNA sequencing and 5’ -> 3’ capture libraries. This sequencing is now underway. A similar experiment was recently completed for Medicago truncatula, to provide data on another legume grown under comparable conditions. Again, this data will be compared to identical experiments conducted with a variety of other plant species. We expect to have the sequencing results very soon.
At the same time, flowers (open and un-open) from field-grown soybean plants (i.e., Williams 82) were also harvested, RNA prepared and shipped to DOE-JGI. Again, sequencing of this material is occurring at JGI. We continue to grow plants to harvest the other major soybean tissues since surveying these tissues is part of the transcriptome atlas plan. We have in the freezer awaiting shipment to JGI samples representing nine different stages of soybean seed development. Plants were seeded into the field this spring to allow for isolation of pollen, as well as the other flower parts.
In addition to these formal arrangements with DOE-JGI, our lab continues to do RNA-seq analysis on other tissues and this information will become part of the overall soybean transcriptome atlas. We now have RNA-seq data from the following conditions:
• Aphid infected tissues. This work involves a comparison of aphid resistant plants (Rag2) vs sensitive plants (rag2). A paper detailing these results and comparing them to proteomic data from the same samples is now being prepared.
• Soybean roots subjected to abiotic stress. The completed study includes a time course of roots after heat treatment. RNA isolated from roots subjected to drought stress will be sequenced soon.
• Soybean leaves after treatment to induce innate immunity, again over a time course. These same samples have been sequenced for analysis of small RNAs.
• Soybean roots treated with the Bradyrhizobium japonicum lipo-chitin nodulation factor. This is the key symbiotic signal involved in inducing soybean nodulation.
Collectively, all of the samples sequenced in year one exceeded our goal of 30 different conditions. We are now proceeding aggressively to continue the sequencing for year two of the project.