2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Analyze mRNA and storage oil profiles of cotyledons over the course of seed maturation to infer biological networks underlying soybean seed oil composition and content, and to predict their key gene candidates.
Objective 2: Analyze small RNA profiles of cotyledons over course of the seed maturation, and further predict small RNA candidates regulating soybean seed oil composition and content.
1b.Approach (from AD-416):
Objective 1: Soybean oil synthesis and deposition occur mainly in cotyledons and are differentially regulated over the course of seed maturation. The oil synthesis and deposition are accomplished through the concerted activities of many gene products and biological pathways that are primarily regulated at transcription levels. Gene expression patterns change very quickly over the course of an organism’s evolution if it is not subjected to functional constraints. Although it is not a universal rule, evidence suggests that expression patterns of many genes are intended to co-evolve with their biological functionalities. The co-evolution is reflected by a correlation of gene expression pattern with the related biological functions, and co-expression of functionally related genes such as those that encode proteins that reside in the same metabolic or signal pathways, or in the same cellular complexes under a variety of biological conditions (Stuart et al., 2003; Wei et al., 2006). The expression pattern correlation is widely used as criteria to predict biological functions of genes, functional relatedness between genes, and gene regulatory networks (Horan et al., 2008). We have examined transcriptomes and storage lipid profiles of cotyledons at six distinct developmental stages over the course of seed maturation.
Objective 2: We have conducted deep sequencing of small RNA populations in the same RNA preparations used for transcriptome analysis. It is believed that miRNAs function as master regulators in gene regulatory networks underlying diverse biological processes in Arabidopsis. However, much less is known about soybean miRNA species and their accumulation patterns over the course of seed maturation. As part of our effort to delineate regulatory networks and identify key genetic components controlling oil composition and content, we will conduct genome-wide characterization of small RNAs, particularly miRNAs, in cotyledons over the course of seed maturation. We will use a bioinformatic approach to analyze the small RNA sequences we have conducted to discover miRNA species and their accumulation patterns. MiRNA species recognize their target mRNA by high sequence complementation, and function mainly as suppressors of the accumulation of the target mRNA by directing the degradation of its functional target mRNAs in plants. Sequence complementarities and negative correlation of mRNA and miRNA accumulation patterns should offer a more effective approach to identify the functional target genes, and can be used to delineate the topology of small RNAs in the TF networks inferred in Objective 1. The miRNA species that locate in TF networks enriched with oil related genes or target oil related genes would be strong candidates for future investigations for oil composition and content improvement.
Production of storage oil in seeds requires concerted activities of many genes and biological pathways over the course of seed maturation. However, lack of knowledge on the intricate biological network and the availability of its key regulatory components has been a bottleneck in the effective application of both breeding and biotechnological approaches for soybean oil quality improvement. To address this problem, we determined profiles of message RNAs, small RNAs, DNA methylation, and major lipid species in soybean cotyledons (soybean cv. Jack) over the course of seed maturation. Having developed and applied a variety of bioinformatic strategies and tools, we identified the genes and biological pathways that are differentially regulated over the course of seed maturation, and inferred a set of transcription factor networks underlying soybean seed maturation. The regulatory networks potentially regulating oil quality traits were also identified. We identified and characterized in detail a set of miRNAs expressed in soybean cotyledons. The research provided great insight into molecular regulatory mechanisms underlying seed maturation and oil qualities at the gene and systems level. The project expired on 3/25/2013.
Introns are preferential sites to evolve novel miRNA genes in plants. Micro-ribonucleic acids (miRNAs), small RNA molecules that control which genes are expressed or repressed in plant tissues, are an emerging class of genetic control agents that regulate a variety of physiological and developmental processes in both plants and animals. In animals a large number of the genes that produce these small regulatory molecules (miRNAs) are found within in the introns of genes. Introns are DNA sequences within a gene that do not code for proteins (the final product of the gene) and are removed from the gene transcript (an RNA molecule that directs the synthesis of the protein) prior to protein synthesis. However, in plants only a few plant miRNA genes have been discovered in gene introns. By mapping mRNA genes, discovered in our analysis of soybean seed miRNA content, we discovered 38 soybean miRNAs that reside in gene introns. Our sequence analysis also indicated that all 38 miRNAs appeared relatively recently within the evolution of the soybean genome. The expression of these miRNAs was highly correlated with the expression of the genes within which they reside. The research indicated that introns are preferred sites for the evolution of novel miRNAs in plants, as appears to be the case in animals. Knowing where to look for novel miRNA genes with plant genomes will greatly facilitate their discovery and knowing that the genes within which they reside influence their expression will allow us to develop novel markers for use in soybean breeding strategies to improve seed quality.
Illustration of miRNAs and their underlying regulatory networks in soybean cotyledons. Micro-ribonucleic acids (miRNAs), small RNA molecules that control which genes are expressed or repressed in plant tissues, are an emerging class of genetic control agents that regulate a wide range of biological processes. Of prime interest to soybean researchers is if these new genetic regulators (miRNAs) control the synthesis and storage of oils and proteins found in the soybean cotyledons, the main site within soybean seeds of these important commodities. However, little is known about what miRNAs accumulate in soybean cotyledons during the stages where these compounds are made and stored during seed development, and how they may regulate these processes. ARS scientists in St. Louis, MO sequenced 292 million small RNA molecules isolated from soybean cotyledons and determined that a total of 243 miRNA genes were expressed in these tissues. The cotyledon miRNAs were predicted to regulate 1061 genes. By applying a computational model, the regulatory networks within which these mRNAs control the expression of the target genes in cotyledons was inferred. This comprehensive characterization of miRNAs and their regulatory networks will open up new avenues and strategies for developing new soybean varieties that have improved oil and protein qualities that benefit both producers and consumers.