Location: Weed and Insect Biology Research
2024 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: Samples collected from replicated field plots of sunflower grown under inter- or intra-specific competition with alfalfa or natural weed populations in Minnesota, and North and South Dakota have been analyzed for nutrient content and used to develop Ribonucleic Acid sequencing (RNAseq) libraries. In Minnesota and North Dakota, sunflower was planted at 72- and 152-centimeter row spacings and interseeded with or without alfalfa. In South Dakota, sunflower was planted at two times the normal planting rate for intra-specific competition studies, or interseeded with natural weeds for inter-specific competition. Leaf and root tissue of two sunflower plants per replicated treatment were collected three times during reproductive development (R1 – R8). Treatments impacted the growth of sunflower plants relative to the controls in South Dakota, but sequence data from intra-specific competition (sunflower-sunflower) or inter-specific competition (sunflower-natural weed populations) only identified one gene of unknown function that was differentially expressed between treatments. Thus, it was impossible to develop any testable hypotheses on that data alone. Analysis of RNAseq data from samples collected in Minnesota and North Dakota related to intra- (sunflower-sunflower) and inter-specific (sunflower-alfalfa) competition are still being analyzed.
Objective 1B: A medium maturity sunflower hybrid (Falcon) and a corn inbred (B73) were planted in pots and grown under greenhouse conditions. For intra-specific completion, 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, or four canola plants, or four amaranth 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 (vegetative stage 6) stage of development. At that point, root-to-root contact was re-established by removing the interfering 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 interfering plants in cone-tainers from the potted soil, but then replacing them 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. Samples collected from intra- and inter-specific competition studies were used to develop RNAseq libraries.
Analysis of the sequence data from the corn vs canola study identified consistent sets of differentially expressed genes. Gene set enrichment analyses identified over-represented ontologies associated with oxidative stress signaling throughout the time of weed exposure, with additional ontologies associated with nitrogen use and transport, and abscisic acid (ABA) signaling, with defense responses being enriched at later time points. Enrichment of promoter motifs among the differentially expressed genes indicated over-representation of sequences known to bind with FAR1, several AP2/ERF transcription factors and others. Likewise, co-expression networks were identified using Weighted-Gene Correlation Network Analysis (WGCNA) and Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) algorithms. The WGCNA highlighted potential roles of several transcription factors (e.g., MYB 3r-4, TB1, WRKY65, CONSTANS-like5, ABF3, HOMEOBOX 12), among others. Several specific proteins involved in ABA signaling were also highlighted as being important for the initiation of the early maize responses to weeds. The SC-ION algorithm highlighted potential roles for NAC28, LOB37, NAC58, and GATA2 transcription factors, among many others. A manuscript describing this work has been published.
The nature of above- and below-ground weed-generated signals also identified a set of differentially expressed genes in corn responding to above- or below-ground signals generated by interfering canola plants. Gene set enrichment analysis indicated that exposure to above-ground signals resulted in induction of oxidative stress responses in the roots, while exposure to below-ground signals surprisingly altered genes associated with photosynthetic processes in the roots and leaves. No consistent response was observed in the leaves of corn exposed to the above-ground signals, but photosynthetic processes were disrupted in the leaves when corn was exposed to the below-ground signals. Network analyses identified clusters of coordinately regulated genes involved in photosynthetic processes and defense-related responses that were enriched among the differentially expressed genes. Multiple transcription factors were differentially expressed in roots or leaves in response to either above or below-ground signals that implicated developmental reprograming and stress responses in corn exposed to the specific weed-generated signals. A manuscript presenting these results is under review.
Objective 2A: Transgenic corn plants containing a reporter gene (Red3 - detected by red fluorescence) turned on by a weed-inducible promoter, isolated from the corn DC1 gene, were grown in a greenhouse for self-pollination and used to identify homozygous lines from T2 seed. The results indicated weed inducibility of the Red3 reporter gene in two independent homozygous transgenic lines, but the response, though consistent, was not significant. Surprisingly, the homozygous lines carrying a NahG gene (a gene coding for salicylate hydroxylase) under control of the putative transgenic promoter was down-regulation when grown with weeds. Additional work will be needed to understand these observations.
Objective 2B: Salicylic Acid (SA) analysis of corn samples collected from field plots in competition with alfalfa or weeds, or under greenhouse conditions in competition with rapeseed, was completed in collaboration with Clemson University and the ARS, Potato Physiology Lab in Fargo, ND. The results indicated that corn growing with weeds under field conditions contained significantly more salicylic acid than the control plants.
Objective 2C: Transgenic corn with NahG under the control of a weed-inducible promoter was obtained. The transgenic plants expressed NahG RNA under control conditions and had enhanced expression under weedy conditions. Homozygous lines containing the NahG gene under control of the weed-inducible DC1 promoter did not show up-regulation of the reporter gene. Additionally, there was no significant difference in response of the transgenic lines to weed presence relative to the wild-type mock-transformed parental lines.
Objective 3A: A Recombinant Inbred Line (RIL) population of camelina has been phenotyped and genotyped. The population was phenotyped for traits such as flowering time, days to maturity, seed yield and size, and freezing tolerance in response to treatment with or without cold acclimation for 8 weeks. Markers mapped to the camelina genome were used to identify Quantitative Trait Loci (QTL) on chromosomes associated with these traits. QTLs for flowering time and freezing tolerance on chromosome 8 and 13 were consistently observed and covered a region of the chromosome containing a gene know as FLOWERING LOCUS C (FLC). Additional loci on chromosomes 16 and 18 have either FLC-like genes such as MAF3 (MADS AFFECTING FLOWERING 3) or genes known to interact with FLC. Manuscripts related to this work are under peer-review, and a manuscript describing homozygosity mapping to identify a region on chromosome 11 associated with freezing tolerance has been published. Abstracts related to QTLs for seed characteristic of the RILs have also been accepted for presentation at multiple international meetings. Work continues to develop constructs that cause Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mutations in each of the three FLC genes, and in the multiple receptor-like kinase genes in transformed camelina.
Objective 3B: Constructs for canola genes (SENSITIVE TO FREEZING 2; SFR2, and VERNALIZATION INDEPENDENCE 3; VIP3) were developed and gene-editing technology and over- or under-expression are being used to characterize their roles in transgenic plants. Sequencing the coding regions of VIP3 from two canola lines with divergent deacclimation rates confirmed a deletion in the VIP3 gene of the slow-deacclimating genotype. Likewise, mutation of VIP3 causes reduced deacclimation rates in Arabidopsis and RNAseq analysis indicated that several COLD-REGULARED (COR) genes had higher expression in mutant lines than in the wildtype Arabidopsis following deacclimation. However, no genes were differentially expressed in mutants relative to the wildtype following acclimation. Further analysis with Assay for Transposase-Accessible Chromatin (ATAC) sequencing revealed a slight but insignificant peak of open chromatin in the COR promoters of the mutant genotypes relative to the wild type. Further analysis of the COR sequences did not suggest differential RNA decay in the mutant genotype relative to the wild type. Work continues on transforming winter canola germplasm with mutants of SFR2 and VIP3.
Objective 3C: A series of constructs designed to turn off the two weed-inducible PHYTOCHROME INTERACTING FACTOR (PIF3) genes or all six of the PIF3 genes from soybean has been completed. These constructs were confirmed by sequencing and CRISPR constructs designed to specifically knock out each of the weed-inducible PIF3 genes were contracted for transformation into soybean and transgenic seeds are expected by early 2025.
Accomplishments
1. Identifying signaling mechanisms involved in weed-induced crop yield loss. Crops perceive weed-generated signals through changes in light quality, and through volatile and soil-soluble chemicals. To mitigate weed-induced yield losses, ARS scientists in Fargo, North Dakota, conducted time course studies to understand how weeds impact genes and growth of corn through soil-soluble signals. These studies identified over-represented gene ontologies associated with oxidative stress signaling throughout the time of weed exposure. Gene ontologies associated with nitrogen use and transport, and abscisic acid (ABA) signaling were also observed, with defense responses enriched at later time points. Many of these genes were over-represented with sequence motifs known to bind with proteins such as FAR1, and several AP2/ERF transcription factors that regulate gene expression. This research provides a valuable first step for identifying potential mechanisms for mitigating weed-induced crop yield losses.
2. Identifying regions of the Camelina sativa genome associated with important agronomic traits. Breeding for desired agronomic traits is critical for improving cropping systems. ARS scientists in Fargo, North Dakota, Clay Center, Nebraska, and Stoneville, Mississippi, and scientists at North Dakota State University collaborated to sequence and identify regions of the genome associated with freezing tolerance, flowering time, seed size, and oil profiles in a population of camelina developed from crossing a winter and spring biotype. Sequencing of these camelina genomes identified specific markers within the 20 assembled chromosomes associated with these important agronomic traits. This work also improved the assembly of camelina reference genomes and helped to identify candidate genes associated with differences observed for these important agronomic traits. These outcomes will assist breeders to improve important agronomic traits in oilseed cash crops used for developing cropping systems suitable for colder climates and soils.
3. Identifying molecular mechanisms for improving freezing tolerance in canola. Acclimating canola plants to low temperatures improves freezing tolerance but short periods of warm temperatures in late fall and early spring can cause deacclimation and loss of freezing tolerance. ARS scientists in Fargo, North Dakota, identified a canola gene known as VERNALIZATION INDEPENDENCE 3 (VIP3) that, when mutated in the model plant Arabidopsis thaliana, prevents deacclimation. A deletion in this gene was identified in deacclimation insensitive canola. The mechanism by which VIP3 reduces deacclimation rates indicated that COLD-REGULATED (COR) genes are expressed longer under deacclimating condition in VIP3 mutant genotypes than in wildtype Arabidopsis. This research confirmed the role of VIP3 as the first plant gene shown to reduce deacclimation rates, furthered our understanding of the deacclimation process, and provides breeders with knowledge for improving freezing tolerance in crops suitable for colder climates and soils and for combating the impact of climate change.
4. The plant hormone salicylic acid acts as a signaling mechanism in weed-induced yield loss. Changes in gene expression of corn in response to weed interference implicated salicylic acid as a probable signal hypothesized to cause reduced growth and yield in corn. ARS scientists in Fargo, North Dakota, tested this hypothesis by examining levels of salicylic acid in multiple varieties of corn plants growing with or without weeds. The results indicated that salicylic acid levels were significantly greater in corn growing in the presence of weeds than in corn growing in weed-free conditions. This work supported the hypothesis that weed-induced salicylic production is one of the primary signals responsible for yield and growth reduction in corn.
Review Publications
Hoque, A., Anderson, J.V., Rahman, M. 2024. Genomic prediction for agronomic traits in a diverse flax (Linum usitatissimum L.) germplasm collection. Scientific Reports. 14. Article 3196. https://doi.org/10.1038/s41598-024-53462-w.
Dogramaci, M., Sarkar, D., Datir, S., Finger, F., Shetty, K., Fugate, K.K., Anderson, J.V. 2024. Methyl jasmonate and 1,4-dimethylnaphthalene differentially impact phytohormonal and stress protective pathway regulation involved in potato tuber dormancy. Postharvest Biology and Technology. 213. https://doi.org/10.1016/j.postharvbio.2024.112931.
Sthapit Kandel, J., Talukder, M.I., Shaikh, T., Horvath, D.P., Li, X., Anderson, J.V. 2024. Identification of quantitative trait loci for flowering time in a Camelina biparental population developed from winter- and spring-type parents. Industrial Crops and Products. 220. Article 119259. https://doi.org/10.1016/j.indcrop.2024.119259.