Location: Sunflower and Plant Biology Research
2018 Annual Report
Objectives
Objective 1: Identify, at the genome and physiological levels, plant-plant interactions that impact plant growth and lead to crop yield losses, especially crop-weed interactions that occur during the critical weed-free period, and interactions that occur between the different crops inter-planted in relay cropping systems, such as corn, soybeans, or sunflowers relayed with camelina, ryegrass, or canola. [NP304, Component 2, Problem Statement 2A3]
Sub-objective 1.A: Determine the parameters for evaluating the impacts of winter annual cover crops on corn, sunflower, and Amaranthus spp. productivity.
Sub-objective 1.B: Identify physiological and molecular mechanisms that control interactions between cover crops and corn, sunflower, and Amaranthus spp.
Sub-objective 1.C: Evaluate impacts of candidate genes on cover crop-relay crop and cover crop-weed interactions.
Objective 2: Determine the molecular and physiological mechanisms by which winter annual cover crops suppress weeds in northern temperate agroecosystems, and identify genes that will enhance weed suppression in these crops, such as genes associated with weed-tolerance, cover-crop tolerance, and cold hardiness. [NP304, Component 2, Problem Statement 2A3]
Sub-objective 2.A: Identify genetic markers for improving the weed-suppressing trait of winter hardiness in winter canola and/or camelina varieties.
Sub-objective 2.B: Evaluate the weed-suppressing traits of winter-hardy canola and camelina in the field.
Approach
Weeds are major pests of agro-ecosystems that reduce production of the nation’s food, feed, fiber and fuel crops. The industry-adopted practice of rotating crops with engineered tolerance to a limited set of herbicides continues to put selection pressure on the evolution of herbicide-resistant weeds. As part of a holistic and sustainable approach to managing weeds in temperate agro-ecosystems, we propose to identify cover crop-relay crop interactions to enhance relay crop productivity, cover crop-weed interactions to enhance weed suppression, and identify winter-hardy annual cover crops that suppress weeds in relay cropping systems. In this proposal, winter canola will serve a dual purpose as both a cover crop for evaluating weed suppression, and as a surrogate weed for weed-relay crop interactions. Our model relay cropping system consists of inter-seeding a commodity crop (corn or sunflower) into an established cover crop (winter canola) such that their lifecycles overlap. Currently, no winter-hardy annual broadleaf cover crops are economically suited for weed suppression in relay cropping systems in the upper Midwest (UMW) and Northern Great Plains (NGP). Consequently, the objectives of this project are: (1) elucidating regulatory signals and pathways associated with cover crop-relay crop and cover crop-weed interactions that impact plant productivity, and (2) identifying economically suited winter-hardy broadleaf cover crops that suppress weed establishment in inter-seeded relay crops. These objectives will be accomplished through the use of physiological, molecular, and genomic approaches, with the long-term goals of revolutionizing weed management practices.
Progress Report
Sub-objective 1.A: Collect samples for analyzing transcriptome responses of relay crops and Amaranthus spp. to winter canola.
We have completed development of efficient protocols for studying crop weed interactions under greenhouse conditions. These protocols have been used to assess the phenological responses of crops (corn and sunflower) to varying weed densities, and the phenological response of sunflower to above and/or below ground weed-generated signals. These studies indicated that above-ground signals (light quality, and/or volatile signals) have a limited impact on growth and development of sunflower. Although light quality signals undoubtedly impact sunflower growth when weed density levels are high enough, the bulk of sunflower yield loss due to weeds appears to result from below ground weed-generated signals. Interestingly, application of activated charcoal to the soil partially, but not completely, reduced phenological responses to below ground weed-generated signals – suggesting 1) multiple factors may be involved in this signaling process, and/or 2) increased concentrations of activated charcoal are needed to completely block the weed-generated signals. Transcriptomics data generated from corn and sunflower samples growing under varying weed densities pressure, and from sunflower responding specifically to above and/or below ground weed-generated signals are currently being analyzed. Results from this analysis are expected to identify genes and pathways in crops (sunflower, corn) responsive to weed density conditions, and to specific signals generated by weeds. Further, we are in the process of examining the perception of corn to the below ground weed generated signals, and have also collected root material from corn at the six-leaf stage after 0, 1, 2 ,3, 7, and 14 days following exposure to weeds. Analysis of these timeline data are expected to identify the initial genes and signaling pathways affecting crop’s perception of weed presence.
Because studying crop-weed interactions is more difficult in large, genetically complex species such as corn and sunflower, we have developed a protocol for utilizing the model plant Arabidopsis as a more efficient method for testing crop-weed interactions under greenhouse conditions. Arabidopsis makes an excellent system to study crop-weed interactions because 1) it is fast growing, 2) exhibits similar responses to weeds as do sunflower and corn, 3) there are extensive publicly available genetic resources such as T-DNA mutations for almost all known genes, 4) it is easily transformed and manipulated, and 5) it is the most extensively studied plant species with an extensive literature resource base. These tools and resources provide an effective method to study the signaling pathways and genes involved in all the suspected mechanisms for weed-induced reductions in crop yield. So far, we have used this system to test the role of several regulatory and/or signaling genes in interspecies competition studies, which demonstrated that PHYTOCHROME INTERACTING FACTOR (PIF) genes, that have a known impact on plant response to far red enriched light conditions, do not alter the ability of Arabidopsis to respond to weedy conditions. We are also using this model system to test if suspected target signaling pathways previously identified in our studies on corn-weed interactions, such as salicylic acid signaling mutants, show higher levels of weed tolerance.
Sub-objective 2.A: Complete phenotyping and genotyping for winter hardiness in the winter canola diversity panel and report results.
We have completed genotyping and phenotyping, under controlled environments, of the winter canola diversity panel obtained from Kansas State University and results from this work have been presented at the American Society of Plant Biologists annual meeting in 2018. Field phenotyping for winter survival of our most promising winter canola accessions is ahead of schedule. Based on our phenotyping studies conducted under controlled environments, we chose the 5 most freezing tolerant accessions from our winter canola diversity panel. The seeds were planted in two consecutive years on either September 7, 2016 or September 5, 2017. Results obtained for overwinter survival in two replicated field plots were less than 30% in year 1 and 0% in year 2. Although these are discouraging numbers, altering several factors may increase these survival rates. We have observed that later fall planting dates increased winter camelina survival compared to late summer and early fall planting dates, and that our smaller winter canola and camelina plants survived winter better than larger ones. These results suggest that planting date and/or developmental maturity may play a critical factor in winter survival of Brassicaceae winter oilseed cover crops. Thus, in the fall of 2018, we will field plant our winter canola accessions at two different planting dates starting in early September and again in late September. Because smaller plants have been noted to have better winter hardiness than larger plants, and our winter canola planted in early September of 2016 and 2017 were relatively large or “vegetatively mature”, we hypothesize they may have been less winter hardy.
Another factor that may be critical to winter survival in northern climates is associated with established USDA winter hardiness zones. Our initial winter diversity panel was obtained from a Kansas State breeding program, which is a region of the U.S. classified as zones 6a-6b (-23.3 to -17.8 C). Thus, germplasm bred for this winter hardiness zone may not be practical for the winter hardiness zones of North Dakota, which are classified as zones 3b-4a (-37.2 to -31.7 C). To overcome this potential issue, we have obtained an additional 222 accessions of winter canola from the Genebank Gatersleben of the Leibniz Institute of Plant Genetics and Crop Plant Research in Gatersleben, Germany. This collection of seed represents germplasm obtained from more northerly areas of Europe including Belgium, Denmark, Germany, Ireland, Lithuania, the Netherlands, Poland, Russia, Sweden, and UK. Seeds from this collection have been planted in Fargo, North Dakota to increase seed production for phenotyping studies related to freezing tolerance and we have also genotyped the population using genotyping-by-sequencing technology. In the fall of 2018, bulked seed from this collection will be field planted in Fargo, North Dakota on September 5 and September 30 and any plants showing winter survival will be bagged for seed collection and further analyses. Because these additional populations were collected from more northerly climates of Europe, we hypothesize they may exhibit greater freezing tolerance compare to our current diversity panel from Kansas State.
Based on results obtained through supplemental grant funding, we have determined that brief warm spells in the late fall through winter and in the early spring can result in cold deacclimation - leaving winter canola vulnerable to subsequent return of freezing conditions. To elucidate threshold conditions leading to cold deacclimation, we tested five accessions with varying deacclimation responses. These studies indicated that as few as 3 days at temperatures greater than 10 C resulted in near complete deacclimation. However, temperatures at 10 C result only in partial deacclimation, regardless of how long the plants remain at 10 C. Our interpretation of the data is that the threshold temperature critical for cold deacclimation occurs at between 5-10 C. This information should allow growers to assess the likelihood of damage to their winter canola based on overwinter weather conditions and take necessary actions.
Sub-objective 2.A: Initiate GWAS analysis on winter canola diversity panel.
Winter canola produces greater yields than spring canola. However, yearly canola acreage is limited due to its inability to withstand harsh winters of the Northern Great Plains. We conducted a genome-wide association study (GWAS) using a previously genotyped diversity panel containing 407 accessions of winter canola. Results from this analysis identified some significant loci that appear to be associated with freezing tolerance. For example, within the associated loci we identified several candidate genes including a TIR-NBS-LRR gene on Chrm A05, previously implicated in freezing tolerance in other species, and a SALT OVERLY SENSITIVE 3 gene on Chrm C07 previously implicated in salt tolerance.
To provide breeders the ability to introgress superior genes for reduced cold deacclimation responses into elite germplasms and increase freezing tolerance of winter canola, we used our winter canola diversity panel to identify genetic loci that can alter the cold deacclimation response. We identified three loci showing high association with cold deacclimation responses in winter canola. GWAS identified loci associated with cold deacclimation in winter canola on Chromosomes ANN Random, CO6 Random and CNN Random. Potential candidate genes located within these loci included a previously uncharacterized DNA binding protein (COLD-REGULATED NUCLEIC ACID BINDING/REPAIR PROTEIN) in Arabidopsis, a PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 3, a LEUCINE RICH REPEAT KINASE, a cold regulated ARGONAUTE 3-like gene, and a Jumonji-like (EARLY FLOWERING 6) gene involved in chromatin modifications in response to environmental conditions, among other potential candidate genes. These observations have allowed us to construct testable hypotheses regarding the role of these genes in the cold deacclimation process.
Accomplishments
1. Identification of a mutation associated with floral regulation in Camelina sativa. Winter-annual biotypes of Camelina require a prolonged cold treatment to induce flowering, whereas summer-annual biotypes do not. Research directed towards identifying genetic factors controlling flowering in Camelina led ARS scientists in Fargo, North Dakota to discover a mutation that occurs at a greater frequency in a key floral regulatory gene of summer-annual compared to winter-annual biotypes. This mutation is predicted to enable flowering in summer-annual biotypes without a cold treatment, and has been used to develop a marker for distinguishing summer- and winter-biotypes early in seedling development. This discovery provides new knowledge for manipulating flowering time, which is an important trait for development of double cropping systems.
2. Identifying genes of Camelina sativa that control winter hardiness. Winter biotypes of Camelina regularly survive winter conditions experienced in North Dakota, whereas summer biotypes of camelina, and winter canola, are generally freezing sensitive to temperatures experienced in northern regions of the U.S. ARS scientists in Fargo, North Dakota analyzed changes in gene expression in response to cold temperatures in a winter- and a summer-annual biotype of camelina. Several novel genes were identified that may allow winter camelina biotypes to survive the harsh winter conditions experienced in North Dakota. These findings suggest that as few as two genes may regulate freezing tolerance. Identifying these cold-tolerance genes in camelina could provide tools for increasing winter hardiness in closely related species, such as canola, and help expand canola production in the U.S.
3. Planting date of winter oilseed cover crops affects winter survival in northern climates. Winter-hardy oilseed cover crops, such as Camelina sativa, provide value-added ecosystem services and double cropping options in northern regions of the U.S. The ability of these winter oilseed cover crops to provide maximum ecosystem services is dependent on their winter survival. ARS scientists in Fargo, North Dakota, in collaboration with scientists from North Dakota State University, demonstrated that planting date of winter biotypes is critical to the overwinter survival of Camelina sativa. Results from this collaborative field study conducted in Fargo, North Dakota indicated that seeds planted in mid- to late-September had the best overwinter survival, spring stand establishment, and yield. This information will provide land managers and extension personnel with the knowledge for making appropriate planting date decisions and recommendations.
4. Identification of signals regulating crop-weed interactions. Crops are proposed to detect weeds though altered light quality, through soil soluble chemical signals, and via volatile signals. ARS scientists in Fargo, North Dakota developed new approaches for separating these signals to determine how crops detect weeds and, more precisely, determined the developmental and physiological pathways that are activated when crops detect weeds. These protocols indicated direct root-to-root contact between the crop and weeds was required for maximum yield loss, regardless of nutrient and water availability. Identifying the signaling mechanisms and plant responses that allow crops to detect weeds could one day lead to the development of crops that are blind to competition, reduce herbicide use, and promote acceptance of cover crop and double cropping systems.
5. A specific weed-induced chemical defense signal in corn. Yield losses due to crop-weed interactions are caused by the costs of defense responses that are activated in crop plants, rather than direct competition with weeds for resources. With collaborators at South Dakota State University, ARS scientists in Fargo, North Dakota identified the involvement of a plant hormone-regulated gene pathway that is activated when weeds are present early in the growing season. Thus, inhibiting this pathway during the early growing season could make crops more tolerant of weeds and/or cover-crops. If successful, this approach will provide growers with a wider developmental window for weed control without loss of yield.
6. Developing Arabidopsis as an efficient model system to study plant-plant interactions. Studying crop-weed interactions is often difficult in genetically complex species such as corn and sunflower. ARS scientists in Fargo, North Dakota have demonstrated that Arabidopsis, the classic plant model, exhibits responses similar to crops under weed pressure. This system has been used to test the role of several regulatory and/or signaling genes suspected of controlling crop-weed interactions, such as genes known to impact plant response to overcrowding, and genes regulating plant defense responses. This model system provides a rapid method for functionally testing genes identified in crop-weed interaction studies.
7. Enabling the development of elite canola germplasm with increased winter hardiness. Short warm spells in the late fall and early spring often result in cold deacclimation, leaving crops such as winter canola vulnerable to subsequent return of freezing conditions. Identifying genes that prevent cold deacclimation in response to short periods of warm temperatures is a critical need for improving winter hardiness in winter canola. ARS scientist in Fargo, North Dakota determined the temperature thresholds and duration needed to initiate cold deacclimation and then used a population of winter canola varieties to identify chromosomal regions associated with the cold deacclimation response. Several genes known to be responsive to cold or involved in chromatin modifications in response to environmental conditions were identified. Manipulation of elite winter canola germplasm with these candidate genes will allow breeders to test their functional activity in enhancing winter hardiness.
Review Publications
Horvath, D.P., Bruggeman, S., Moriles-Miller, J., Anderson, J.V., Dogramaci, M., Scheffler, B.E., Hernandez, A.G., Foley, M.E., Clay, S. 2018. Weed presence altered biotic stress and light signaling in maize even when weeds were removed early in the critical weed-free period. Plant Direct. Available: https://onlinelibrary.wiley.com/doi/abs/10.1002/pld3.57.
Carvalho, L.J.C.B., Anderson, J.V., Chen, S., Mba, C., Dogramaci, M. 2018. In: Waisundara, V., editor. Cassava. Domestication syndrome in cassava (Manihot esculenta Crantz): Assessing morphological traits and differentially expressed genes associated with genetic diversity of storage root. Rijeka, Croatia. InTech. https://doi.org/10.5772/intechopen.71348.
Carvalho, L.J.C.B., Filho, J.F., Anderson, J.V., Figueiredo, P.G., Chen, S. 2018. Storage root of cassava: Morphological types, anatomy, formation, growth, development and harvest time. In: Waisundara, V., editor. Cassava. Rijeka, Croatia. InTech. https://doi.org/10.5772/intechopen.71347.
Horvath, D.P., Patel, S., Dogramaci, M., Chao, W.S., Anderson, J.V., Foley, M.E., Scheffler, B., Lazo, G., Dorn, K., Yan, C., Childers, A., Schatz, M., Marcus, S. 2018. Gene space and transcriptome assemblies of leafy spurge (Euphorbia esula) identify promoter sequences, repetitive elements, high-quality markers, and a full-length chloroplast genome. Weed Science. 66(3):355-367. https://doi.org/10.1017/wsc.2018.2.
Anderson, J.V., Horvath, D.P., Dogramaci, M., Dorn, K.M., Chao, W.S., Watkin, E.E., Hernandez, A.G., Marks, M.D., Gesch, R. 2018. Expression of FLOWERING LOCUS C and a frameshift mutation of this gene on chromosome 20 differentiate a summer and winter annual biotype of Camelina sativa. Plant Direct. 2:1-14. https://doi.org/10.1002/pld3.60.
Maroli, A.S., Gaines, T.A., Foley, M.E., Duke, S.O., Dogramaci, M., Anderson, J.V., Horvath, D.P., Chao, W.S., Tharayil, N. 2018. OMICS in weed science research: A perspective from genomics, transcriptomics and metabolomics approaches. Weed Science. https://doi.org/10.1017/wsc.2018.33.
Zhanga, L., Loua, J., Foley, M.E., Gu, X.-Y. 2017. Comparative mapping of quantitative trait loci for seed dormancy between tropical and temperate ecotypes of weedy rice (Oryza sativa L.). Genes, Genomes, Genetics. 7(8):2605-2614. https://doi.org/10.1534/g3.117.040451.
