Recipient Organization
VIRGINIA STATE UNIVERSITY
(N/A)
PETERSBURG,VA 23803
Performing Department
Dept Of Agricultural Research
Non Technical Summary
The world's population is expected to reach 9.7 billion in 2050. To feed the ever-increasing human population, global crop production may need to be increased by at least 50% by 2050. Traditional breeding played an important role for dramatic yield increases during the Green Revolution. However, annual yield gains for several major staple food crops have been plateaued in the past two decades. Increasing crop productivity has become the most challenging issue facing agricultural scientists and new technological solutions are needed to meet the global demand for crop improvement. One such strategy that may hold untapped potential for sustainable yield increases is to improve crops' photosynthetic efficiency. Another potential strategy is to explore wild species, and identify novel, but often neglected genes that can be used for the enhancement of crops' resilience to various environmental stresses, as well as the improvement of yield and quality. The long effort in PI's laboratory is to explore Purslane (Portulaca oleracea), a wild species, for its potential use in crop improvement. Recently, we cloned the DRM2 homologous gene from Purslane (PoDRM2). Interestingly, except the known conserved domains, PoDRM2 also contains a unique clfB domain at its N terminal that does not exist in all other available DRM2 homologs. Overexpression of PoDRM2 in Arabidopsis significantly increased biomass and leaf starch accumulation. In addition, the PoDRM2 transgenic line significantly increased chlorophyll content suggesting a role of PoDRM2 in regulating photosynthesis. DRM2 acts as major DNA methyltransferase and is essential for RNA-directed de novo DNA methylation of cytosine. Based on our preliminary research and the role of DRM2 in DNA methylation, we hypothesize that PoDRM2 regulates starch accumulation and biomass production through epigenetic regulating plant photosynthesis. To test this hypothesis, we will generate transgenic soybean and corn lines overexpressing PoDRM2 and examine their photosynthesis-related traits. In addition, we will also systemically investigate molecular mechanisms and gene networks regulating PoDRM2 mediated epigenetic control of plant photosynthesis. This project addresses the fundamental issue of crop production-crop photosynthesis, and would generate basic knowledges on epigenetic control of plant photosynthetic efficiency.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long term goal is to enhance US capacity to produce more food, feed and fibers by exploring genetic resources that are underutilized. To achieve this goal, we propose here to test the potential of PoDRM2 from Purslane, a wild species, in improving crop starch accumulation and photosynthetic efficiency, and to elucidate mechanisms of this PoDRM2-mediated epigenetic regulation of starch accumulation and photosynthetic efficiency. There are three (3) specific research objectives for the current proposal. Objective 1: Translating PoDRM2-mediated epigenetic control of biomass and starch accumulation observed in Arabidopsis into maize and soybean, and their physiological characterization; Objective 2: Dissecting molecular function of PoDRM2 through integrated transcriptome and DNA methylation analysis; and Objective 3: Functional characterization of clfB domain on PoDRM2 protein and identification of its physical interacting partners in Arabidopsis.
Project Methods
To implement these specific objectives, we will first create transgenic lines overexpressing PoDRM2 through an agrobacterium-mediated gene transformation approach for both corn and soybean respectively. The transformation will be conducted through a service at the Wisconsin Crop Innovation Center at University of Wisconsin. Independent homozygous transgenic corns and soybeans will be bred at the greenhouse of Virginia State University. At least two independent homozygous transgenic lines for both corn and soybean will be selected for further characterization. The selected transgenic lines, together with the segregating wildtype lines will be subject to a series of experiments conducted to examine physiological parameters related to photosynthesis and starch accumulation. Parameters investigated include plant morphological traits (leaf number, plant height, stem diameters, and leaf areas) and total biomass, stomata number, leaf starch content, chlorophyll content, net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate. We will also examine the molecular functions of PoDRM2 through integrated transcriptome and DNA methylation analysis. Both transgenic soybean and corn lines and their wildtype controls will be subjected to 1) DNA methylation analysis through RRBS-Gene Methylation method; and 2) transcriptome analysis. For RRBS, we will choose restriction digestion enzymes such as MseI and CviQI, that are reported working for plant species-based RRBS. The sequencing will be conducted through service and the genomics data will be analyzed on CyVerse, a platform supported by NSF and free for use of genomic analysis. RRBS methylation data and transcriptome data will be integrated to determine the correlations between differential methylation in genes, promoters or transposable elements with gene expression data. Furthermore, we will characterize functions of clfB domain and identify its physical interacting partners. This set of experiments will be conducted in Arabidopsis. We will first generate transgenic lines 1) overexpressing truncated PoDRM2 protein only containing the first 180 aa (clfB domain); 2) overexpressing the truncated PoDRM2 only containing the aa from position 181 to 617 (containing all domains except clfB). The start codon and stop codon will be introduced when necessary. The two constructs will be transformed into Arabidopsis ecotype Col-0 to breed to homozygous lines. These transgenic lines, together with a full length PoDRM2 transgenic line and Col-0 will be subjected for phenotypic evaluationed as described above. In addition, we will conduct a RNA-seq analysis at the two-week-old seedling stage to identify DEGs between the genotypes. By systemic analysis of different sets of DEGs from different comparative pairs, we will pinpoint what genes, gene networks and pathways are affected by clfB domain. Furthermore, we will also conduct whole genome bisulfite sequencing analysis among genotypes to examine the effect of clfB domain on genome-wide DNA methylation profiles. Such analysis would help to elucidate if clfB domain is required for PoDRM2-mediated DNA methylation, or whether clfB plays some other roles in addition to DNA methylation or clfB just affects a portion of genes that is methylated by PoDRM2. Lastly, we will conduct yeast two hybrid screening using clfB domain as a bait to identify the interacting proteins in Arabidopsis cDNA library we previously established. All identified positive clones will be confirmed following the recommended protocols to eliminate false positive interactions.