Location: Sunflower and Plant Biology Research
2022 Annual Report
Objectives
Objective 1: Determine the nature of inter and intraspecific competition and extent of crop yield loss among relay or double cropped agricultural plant species in comparison to natural weed competition.
Sub-objective 1A: Identify, under field conditions, the genes that are differentially regulated by natural weed populations, cover crops, and intra-specific competition in sunflower (Helianthus annuus).
Sub-objective 1B: Examine the impact of intra-specific competition on sunflower and corn (Zea mays L.) yield loss and gene expression under controlled conditions
Objective 2: Identify genetic or biochemical signals associated with interspecific competition and determine the associated biological mechanisms that can be used as targets for genetic manipulation.
Sub-objective 2A: Create constructs from corn promoters to identify the transcription factor(s) binding sites regulating weed- and/or cover crop-inducible genes.
Sub-objective 2B: Test if changes in salicylic acid levels corresponds to weed perception in corn.
Sub-objective 2C: Utilize the weed inducible promoter from corn to suppress the salicylic acid signaling during weed-crop or crop-cover crop interactions under controlled greenhouse conditions.
Objective 3: Functionally characterize specific targets impacting interspecific competition for genetic manipulation of weed tolerance, winter survival, early maturation, and/or response to bioherbicides.
Sub-objective 3A: Identify winter hardy canola and camelina germplasm that also have an early maturity trait for reducing competition between the cover crop and the relay-crop. As a first step, we will map early maturation Quantitative Trait Loci (QTLs) in a segregating Recombinant Inbred Line (RIL) population of Camelina sativa.
Sub-objective 3B: Determine if the freezing tolerance genes identified from winter camelina will increase freezing tolerance in canola (Brassica napus L.).
Sub-objective 3C: Functionally characterize the weed-induced PIF3 genes in soybean (Glycine max (L.) Merr).
Approach
Integrated weed management (IWM) is considered the most effective approach for managing weeds. In the northern Great Plains, incorporation of winter-hardy crops or cover crops as components of IWM systems are gaining popularity as an approach for managing weeds and the spread of herbicide resistant weeds. However, just like weeds, inter-specific competition with winter crops or cover crops, when used in multi-cropping systems, results in yield losses in major commodities. In this project, multi-cropping refers to fall-planting of oilseed cover crops that overwinter and are terminated or harvested prior to planting a primary summer commodity crop (double-cropping) or a primary commodity crop inter-seeded into the cover crop such that their life cycles overlap (relay-cropping). Factors impacting competition-induced yield losses have only been evaluated in a limited number of traditional multi-cropping systems under field conditions and this gap in knowledge needs to be addressed as new cropping and IWM systems suitable for the northern Great Plains are developed. To generate new knowledge for regionally-appropriate IWM approaches, the goals of this project are to: 1) understand how major commodity crops perceive and respond to inter- and intra-specific competition, 2) identify genes regulating winter survival and early maturity that can be manipulated to improve these traits in winter crops and cover crops, and 3) identify targets for mitigating competition-induced yield losses through breeding or genetic manipulation. Being able to multi-crop major commodities with winter-hardy crops or cover crops without resulting in yield loss, or mitigating weed-induced yield losses in general, would provide new IWM options. Thus, the objectives of this project will address gaps in our knowledge that limit the ability to develop sustainable IWM approaches appropriate for agricultural intensification in the northern Great Plains.
Progress Report
Objective. 1A: Tissue samples collected from field plots of sunflower grown under inter- or intra-specific competition with alfalfa or natural weed populations in North and South Dakota in 2021 have been analyzed for nutrient content and used for developing Ribonucleic Acid sequencing (RNAseq) libraries that are in the process of being sequenced. Field studies were replicated in 2022 at Hickson, North Dakota, Red Lake Falls, Minnesota, and Brookings, South Dakota. At the North Dakota and Minnesota locations, sunflower was planted at two different row spacings (72- and 152- centimeter) and interseeded with or without alfalfa. At the South Dakota location, sunflower was planted at two times the normal planting rate for intra-specific competition studies or was interseeded with natural weeds for inter-specific competition studies. At the V4 (vegetative 4) stage of sunflower development, one treatment at the South Dakota location included removal of cover crops or weeds as competitors of sunflower. At the R1-R8 (reproductive 1 - 8) stage of development, tissue samples were collected three times from healthy leaf and from roots (including soil associated with the rhizosphere) of two sunflower plants per replicated treatment. Samples were frozen and stored for future RNA extraction and analysis of leaf nutrient content. Sunflower plant height, stem diameter, and fresh and dry weights are being recorded at various stages of sunflower development at all locations.
Objective 1B: Data and samples collected from the first replicated study on intra-specific competition of sunflower and corn have been analyzed and tissue samples have been used to develop RNAseq libraries that are in the process of being sequenced. We have completed collecting data for the second replicated study on intra-specific competition, which included a medium maturity sunflower hybrid (Falcon) and a corn inbred (B73) planted in pots and grown under greenhouse conditions. Sunflower and corn were grown in 2- gallon pots, or in 0.5-gallon pots (to determine impact of reduced soil volume). Treatments included 1, 2, or 4 sunflower plants per pot. At 4 weeks of growth, plant height and stem diameter data were collected. At the R4 stage of sunflower development, plant height, stem diameter, and fresh and dry weight of above ground tissue were recorded, and tissue samples were collected from a healthy mid-level leaf and from root tissue of each sunflower replicate per treatment.
For corn, a single corn seedling was grown in a 2-gallon pot with four additional corn plants grown in cone-tainers in the same pot to prevent root-to-root contact and with opaque above-ground cones to prevent light quality signaling between plants until corn was at the V6 stage of development. At that point, root-to-root contact was re- established by removing the corn plants from their cone-tainers and replanting the corn plant back into the soil in half of the pots. The other half were mock treated by removing the corn in cone-tainers from the potted soil, but then replacing the corn and cone-tainers back into the potted soil as they were. Root material was collected at 0, 1, 2, 3, 7, and 14 days from treated and mock-treated plants. Collected samples were used to develop RNAseq libraries, which have been sequenced.
Objective 2A: Constructs (circular DNA used for introducing a modified segment of DNA into plants) which have a reporter gene (Red3 - which is easily detected by red fluorescence) turned on by a weed-inducible promoter (a part of the gene that helps to regulate gene expression) were constructed and subsequently transferred into corn. Multiple corn lines with the transferred constructs were obtained and the promoter worked as expected and expresses the reporter gene under control conditions and shows enhanced expression under weedy conditions. Additionally, the weed-inducible promoter was several likely transcription factor binding sites (specific DNA sequences within the promotor that bind other proteins to help regulate gene expression), which are being characterized using clustered regularly interspaced short palindromic repeats (CRISPR) technology to determine their involvement in the weed-inducibility of genes.
Objective 2B: Corn samples collected from field plots in competition with alfalfa or weeds or under greenhouse conditions in competition with rapeseed have been extracted for salicylic acid (a plant hormone) analysis. Initial High-Performance Liquid Chromatography (HPLC) runs to determine levels of salicylic acid in extracted samples revealed inconsistencies in samples spiked with internal standards. We are currently determining the reason for these inconsistent results, which includes consultation with local experts in HPLC analysis of biological samples. We are testing the use of specialized siliconized vials for injection of salicylic acid standards, which has produced promising results. Once we have resolved the issue with inconsistent results from spiked samples, we will continue with analyzing the extracted samples. Tissue samples (rep 2) from field plot and greenhouse studies have been collected and will be extracted in the fall of 2022.
Objective 2C: Constructs which have NahG (a gene that makes salicylate hydroxylase) and is turned on by the corn weed-inducible promoter was transferred into corn. Multiple corn lines with the construct were obtained and the promoter worked as expected and expresses NahG RNA under control conditions and shows enhanced expression under weedy conditions. These studies are being repeated.
Objective 3A: An F7 Recombinant Inbred Line (RIL) population, consisting of 254 lines, has been phenotyped and genotyped. The population was phenotyped for flowering time, plant height, biomass, days to maturity, seed yield, and freezing tolerance in response to treatment with or without cold acclimation for 8 weeks. Genotyping the RIL population identified over 3700 high quality single nucleotide polymorphism (SNP) markers. A graduate student is mapping the SNPs to the camelina genome and identifying regions of the chromosomes linked to early maturity and freezing tolerance. The student has also completed mapping of selected lines from an F3 RIL population and identified a region of Chromosome 11 containing several cysteine-rich RLK (RECEPTOR-like protein kinase) genes and a receptor serine/threonine kinase gene that are all differentially expressed during cold treatment between the two parental camelina biotypes used to generate the RIL population. Several manuscripts related to phenotyping and genotyping of the camelina RIL population will be submitted in 2022.
Objective 3B: Due to a critical vacancy, a Postdoctoral Research Associate and several graduate students have been recruited to join the project. Although we have not yet definitively identified candidate genes for freezing tolerance from camelina, we have identified several candidate genes associated with freezing tolerance from canola. The Postdoc is working on the design of constructs for a canola gene (SENSITIVE TO FREEZING 2; SFR2) identified from a Genome-Wide Association Studies using a polymerase chain reaction cloning method. CRISPR technology and over-expression of target genes will be applied to characterize the roles of SFR2 in plants. Likewise, a graduate student is working on building constructs to both turn off and over-express VERNALIZATION INDEPENDENCE 3 (VIP3), which is involved in regulating deacclimation processes in canola and arabidopsis. The student has cloned multiple genomic copies of VIP3 from canola varieties with differing deacclimation rates and these clones are currently being sequenced. We are also in the process of confirming the impact of turning off VIP3 on the transcriptome of arabidopsis.
To validate the function of candidate genes in freezing tolerance, we have developed an efficient plant regeneration system using canola hypocotyl segments excised from three different regions (top, medium, and bottom) of 6-, 7-, and 8-day old seedlings. Hypocotyl segments excised from the top region of 8-day old seedlings exhibited the greatest potential for shoot regeneration. A graduate student is working on developing genetic transformation systems for canola using the above-mentioned regeneration system.
Objective 3C: A visiting postdoc created a series of constructs designed to turn off the two weed-inducible Phytochrome Interacting Factor (PIF3) genes from soybean, and another set of constructs that would turn off all six of the PIF3 genes from soybean. These constructs were confirmed by sequencing and are ready to be transferred into soybean.
Accomplishments
1. NDOLA2, a new high-yielding, open pollinated canola variety for the United States. Improving canola production in the United States requires development of breeding lines that are high yielding and carry other agronomic traits desired by the canola industry. ARS scientists in Fargo, North Dakota, and Peoria, Illinois, collaborated with university partners at North Dakota State University to characterize oil quality traits and fatty acid profiles as part of canola variety trials that included a cross made between a European winter- and spring-type cultivar. This collaborative work resulted in the registration and public release of a new high-yielding, open pollinated variety of canola known as NDOLA2. This non-genetically modified spring-type canola provides an important new source for organic markets that is well adapted to North Dakota agro-ecosystems.
2. A candidate gene for increasing freezing tolerance in canola. Winter canola generally produces greater yields than spring canola, but is limited by its inability to withstand the winter conditions experienced in many regions of the northern United States. ARS scientists in Fargo, North Dakota, recently completed a Genome-Wide Association Study conducted on the freezing tolerance of a European population of winter canola and closely related rapeseed varieties. The work identified several candidate genes associated with freezing tolerance, including SENSITIVE TO FREEZING 2 (SFR2) that is known to be involved in the remodeling of certain membranes within plant cells in response to freezing. Transferring these candidate genes from freezing-tolerant lines into elite varieties of canola could help meet the grower and industry goal of increasing canola production in the United States.
3. A gene for improving deacclimation resistance in canola. Global warming is expected to increase fluctuations in winter freeze/thaw cycles, which could cause biennial crops such as canola to rapidly deacclimate and make them susceptible to freezing damage. ARS scientists in Fargo, North Dakota, completed a Genome-Wide Association Study conducted on deacclimation resistance and subsequent freezing tolerance in winter canola varieties. The work identified several candidate genes associated with deacclimation resistance and freezing tolerance, including a gene called VERNALIZATION INDEPENDENCE 3 (VIP3) that is involved with flowering in a closely related species of canola known as arabidopsis. The work further identified a region of DNA surrounding VIP3 in canola and arabidopsis that when altered in arabidopsis increased freezing tolerance. This new knowledge will help scientists to engineer deacclimation resistance and increased freezing tolerance into elite lines of canola, and to meet the grower and industry goal of increasing canola production in the United States.
4. A weed-inducible promoter from corn identified. Reducing the use of herbicides would lower costs for growers and help to protect the environment. Research conducted by ARS scientists in Fargo, North Dakota, identified a gene in corn called DOMAIN-CONTAINING 1 that is up-regulated by the presence of weeds. They further isolated the promotor (a genetic region that turns the gene on) and attached it to a reporter gene. The promotor and reporter gene were then transferred into corn, and it was demonstrated that this promotor increased expression of the reporter gene in the presence of weeds. The identification of this weed-inducible promoter from corn is an important first step for engineering corn to be more resistant to weeds and for dissecting signaling pathways involved in crop-weed interactions.
Review Publications
Mukhlesur, R., Del Rio Mendoza, L., Anderson, J.V., Berhow, M.A., Roy, J., Eriksmoen, E., Ramsey, M., Pradhan, G., Rickertsen, J., Ostlie, M., Hanson, B. 2021. ‘NDOLA-2’, a high-yielding open-pollinated conventional spring type canola in North Dakota. Journal of Plant Registrations. 16(1):124-131. https://doi.org/10.1002/plr2.20189.
Wang, X., Zhang, R., Huang, Q., Shi, X., Li, D., Sao, L., Xu, T., Horvath, D.P., Zhang, J., Xia, Y. 2022. Comparative study on physiological responses and gene expression of bud endodormancy release between two herbaceous peony cultivars (Paeonia lactiflora Pall.) with contrasting chilling requirements. Frontiers in Plant Science. 12. Article e772285. https://doi.org/10.3389/fpls.2021.772285.
Gesch, R.W., Mohammed, Y.A., Walia, M.K., Hulke, B.S., Anderson, J.V. 2022. Double-cropping oilseed sunflower after winter camelina. Industrial Crops and Products. 181. Article 114811. https://doi.org/10.1016/j.indcrop.2022.114811.
Chao, W.S., Li, X., Horvath, D.P., Anderson, J.V. 2022. Genetic loci associated with freezing tolerance in a European rapeseed (Brassica napus L.) diversity panel identified by genome-wide association mapping. Plant Direct. 6(5):e405. https://doi.org/10.1002/pld3.405.
Zhao, X., Zhao, C., Neu, Y., Chao, W.S., He, W., Wang, Y., Mao, T., Bai, X. 2022. Understanding and comprehensive evaluation of cold resistance in the seedlings of multiple maize genotypes. Plants. 11(4). Article 1881. https://doi.org/10.3390/plants11141881.
Anderson, J.V., Neubauer, M., Horvath, D.P., Chao, W.S., Berti, M.T. 2021. Analysis of Camelina sativa transcriptomes identified specific transcription factors and processes associated with freezing tolerance in a winter biotype. Industrial Crops and Products. 177. Article 114414. https://doi.org/10.1016/j.indcrop.2021.114414.
