Location: Plant Science Research2011 Annual Report
1a. Objectives (from AD-416)
Objective 1: Use discoveries from Medicago truncatula, a model legume, root and root nodule genomics to characterize and improve the biological efficiency of symbiotic nitrogen (N2) fixation in alfalfa. Objective 2: Develop and employ RNA interference (i) -mediated gene silencing to identify the functional role of genes involved in phosphorus and nitrogen acquisition and metabolism in root tissues of legumes such as common bean, pea, and lentil. Objective 3: Determine genes regulating protein and oil accumulation in soybean through whole genome transcript analysis and functionally characterize gene candidates.
1b. Approach (from AD-416)
Alfalfa genes important in nitrogen fixation will be identified from the Medicago EST and genome sequencing projects. The sequence of 5'-promoter regions of selected genes will be identified from both alfalfa and M. truncatula. The expression of selected root nodule genes will be silenced through RNA interference. Nodules from plants containing RNA interference will be characterized for nitrogen fixation and nodule development. The Medicago truncatula gene chip will be used to assess global gene expression in alfalfa. BSL-1; Recertified May 19, 2011.
3. Progress Report
Root nodule transporter genes from Medicago, soybean, and common bean were identified, sequenced, and gene silencing constructs transformed into soybean and alfalfa. Transgenic plants containing constructs for over-expression and gene silencing are growing and will be evaluated in 2012. High-throughput RNA-seq experiments were conducted on lupin, common bean, alfalfa, and soybean. White lupin roots subjected to phosphorus stress were evaluated by RNA-seq and a lupin gene index developed. Soybean mutants developed through fast-neutron mutagenesis were evaluated for genome alterations by exome resequencing and comparative genome hybridization. Numerous gene deletions were detected. Phenotype analysis of mutants showed that fast neutron bombardment of soybean seed resulted in plants with altered development, unusual stature, and modified oil and protein content. A database was established for public access to soybean mutant information.
1. Understanding genetic regulation of soybean growth and development. A major objective for the legume community is to develop genomic tools to understand soybean growth, development, and quality. In order to understand genetic regulation of soybean, ARS researchers at St. Paul, Minnesota, in collaboration with State colleagues developed a large population of soybean mutants generated through mutagenesis. Visual phenotype was evaluated on some 20,000 individual M2 fast neutron mutant lines and seed collected from each line. Approximately 250 lines had visual phenotypes. Mutant plant phenotypes include chimeric plants, leaves for flowers, rugose leaves, upright Christmas tree-like stature, no pubescence, and dwarfs. Oil and protein have been evaluated on seed of 15,000 individual M2 and M3 mutant plants. Wide variation was seen for these quality components. Wet lab analysis was completed on seed of 5,000 lines for fatty acid composition. Wide variation was also seen for fatty acid composition. Several lines were selected for further analysis. In collaboration with University of Minnesota colleagues, comparative genome hybridization was completed on 30 individual M2-M3 lines. Genomic deletions in 30 individual lines were mapped to the soybean genome. Deletions range in size from 1.5 kb to sizeable deletions of chromosome arms. Deletions were confirmed by exome resequencing. This research provided 20,000 new mutant soybean lines to the public and a publically accessible website describing the fast neutron mutants was established at SOYBASE.
2. Phosphorus stress in legumes. Although phosphorus (P) is abundant in soils, it is largely unavailable for uptake by plants and is frequently the most limiting nutrient for plant growth and development. ARS researchers in St. Paul, Minnesota, in collaboration with University of Minnesota cooperators conducted next generation high-throughput RNA sequencing on leaves and roots of P-stressed white lupin. Millions of base pairs of white lupin RNA sequence were generated and are being analyzed. A white lupin gene index is being developed. This research identified genes crucial to crop acclimation to P-deficiency that will be useful in selecting plants with improved P-deficiency tolerance.
3. Sugars are required for plant response to abiotic stress. Sugars from photosynthesis have been shown to be integrally involved in the expression of genes that respond to phosphorus, iron, and sulfur deficiency. ARS scientists at St. Paul, Minnesota, have discovered a mutant Arabidopsis plant having extremely high expression of a gene designated sucrose transporter 2 (SUC2). Plants having high expression of SUC2 show a phenomenal increase in growth. Experiments are currently underway to determine how over-expression of SUC2 increases plant growth. These findings appear to have identified a genetic change that significantly increases plant yield and may be useful in crop plant improvement.
4. Plant transporters are required for symbiotic nitrogen fixation and legume seed development. Legumes are important for agriculture because they can symbiotically fix atmospheric nitrogen, thus requiring no nitrogen fertilizer, and because their seeds are rich sources of protein and oil. ARS scientists at St. Paul, Minnesota, in collaboration with colleagues at the University of Minnesota conducted whole genome high-throughput RNA-sequencing on root nodules and developing seed of soybean and common bean. Plant genes expressed only in nodules and seeds were identified and expression levels calculated. Selected genes identified as being crucial to symbiosis and/or seed development were chosen for further analysis. Using biotechnology approaches, the expression of those select transporters will be altered in symbiosis and seed development. Modifying gene expression through conventional breeding and/or biotechnology can enhance nitrogen fixation and seed quality.
Yamagishi, M., Zhou, K., Osaki, M., Miller, S.S., Vance, C.P. 2011. Real-time RT-PCR profiling of transcription factors including 34 MYBs and signaling components in white lupin reveals their P status dependent and organ-specific expression. Plant and Soil Journal. 342(1-2):481-493.