The U.S. Department of Agriculture–Agricultural Research Service (USDA-ARS) has been a leader in weed science research covering topics ranging from the development and use of integrated weed management (IWM) tactics to basic mechanistic studies, including biotic resistance of desirable plant communities and herbicide resistance. ARS weed scientists have worked in agricultural and natural ecosystems, including agronomic and horticultural crops, pastures, forests, wild lands, aquatic habitats, wetlands, and riparian areas. Through strong partnerships with academia, state agencies, private industry, and numerous federal programs, ARS weed scientists have made contributions to discoveries in the newest fields of robotics and genetics, as well as the traditional and fundamental subjects of weed–crop competition and physiology and integration of weed control tactics and practices. Weed science at ARS is often overshadowed by other research topics; thus, few are aware of the long history of ARS weed science and its important contributions. This review is the result of a symposium held at the Weed Science Society of America’s 62nd Annual Meeting in 2022 that included 10 separate presentations in a virtual Weed Science Webinar Series. The overarching themes of management tactics (IWM, biological control, and automation), basic mechanisms (competition, invasive plant genetics, and herbicide resistance), and ecosystem impacts (invasive plant spread, climate change, conservation, and restoration) represent core ARS weed science research that is dynamic and efficacious and has been a significant component of the agency’s national and international efforts. This review highlights current studies and future directions that exemplify the science and collaborative relationships both within and outside ARS. Given the constraints of weeds and invasive plants on all aspects of food, feed, and fiber systems, there is an acknowledged need to face new challenges, including agriculture and natural resources sustainability, economic resilience and reliability, and societal health and well-being.

Modern high-throughput molecular and analytical tools offer exciting opportunities to gain a mechanistic understanding of unique traits of weeds. During the past decade, tremendous progress has been made within the weed science discipline using genomic techniques to gain deeper insights into weedy traits such as invasiveness, hybridization, and herbicide resistance. Though the adoption of newer “omics” techniques such as proteomics, metabolomics, and physionomics has been slow, applications of these omics platforms to study plants, especially agriculturally important crops and weeds, have been increasing over the years. In weed science, these platforms are now used more frequently to understand mechanisms of herbicide resistance, weed resistance evolution, and crop–weed interactions. Use of these techniques could help weed scientists to further reduce the knowledge gaps in understanding weedy traits. Although these techniques can provide robust insights about the molecular functioning of plants, employing a single omics platform can rarely elucidate the gene-level regulation and the associated real-time expression of weedy traits due to the complex and overlapping nature of biological interactions. Therefore, it is desirable to integrate the different omics technologies to give a better understanding of molecular functioning of biological systems. This multidimensional integrated approach can therefore offer new avenues for better understanding of questions of interest to weed scientists. This review offers a retrospective and prospective examination of omics platforms employed to investigate weed physiology and novel approaches and new technologies that can provide holistic and knowledge-based weed management strategies for future.

Leafy spurge (Euphorbia esula L.) is an invasive perennial weed infesting range and recreational lands of North America. Previous research and omics projects with E. esula have helped develop it as a model for studying many aspects of perennial plant development and response to abiotic stress. However, the lack of an assembled genome for E. esula has limited the power of previous transcriptomics studies to identify functional promoter elements and transcription factor binding sites. An assembled genome for E. esula would enhance our understanding of signaling processes controlling plant development and responses to environmental stress and provide a better understanding of genetic factors impacting weediness traits, evolution, and herbicide resistance. A comprehensive transcriptome database would also assist in analyzing future RNA-seq studies and is needed to annotate and assess genomic sequence assemblies. Here, we assembled and annotated 56,234 unigenes from an assembly of 589,235 RNA-seq-derived contigs and a previously published Sanger-sequenced expressed sequence tag collection. The resulting data indicate that we now have sequence for >90% of the expressed E. esula protein-coding genes. We also assembled the gene space of E. esula by using a limited coverage (18X) genomic sequence database. In this study, the programs Velvet and Trinity produced the best gene-space assemblies based on representation of expressed and conserved eukaryotic genes. The results indicate that E. esula contains as much as 23% repetitive sequences, of which 11% are unique. Our sequence data were also sufficient for assembling a full chloroplast and partial mitochondrial genome. Further, marker analysis identified more than 150,000 high-quality variants in our E. esula L-RNA–scaffolded, whole-genome, Trinity-assembled genome. Based on these results, E. esula appears to have limited heterozygosity. This study provides a blueprint for low-cost genomic assemblies in weed species and new resources for identifying conserved and novel promoter regions among coordinately expressed genes of E. esula.

Microarray analysis was used to follow changes in gene expression coinciding with seasonal changes in the dormancy status of crown buds of field-grown leafy spurge. Known cold-regulated genes were induced, and numerous gibberellic acid–responsive genes were down-regulated during the transition from paradormancy to endodormancy. Genes involved in photomorphogenesis were induced during endodormancy. Also, ethylene signaling responses were observed during ecodormancy rather than endodormancy. These results provide additional insights into the signals regulating expression of several genes previously associated with transition from paradormancy to growth in root buds.

Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR, is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of real-time PCR, the major difficulty associated with traditional quantitative or semiquantitative PCR was to ensure that PCR reactions were quantified within the exponential phase of amplification. Real-time PCR alleviates that problem by detecting and quantifying fluorescent signals after each amplification cycle. Additionally, it does not require running gels and thus is able to produce data in 2 to 3 h. Four different types of chemistries, DNA-binding agents (SYBR Green), hydrolysis probes (TaqMan), hairpin probes (molecular beacons, scorpions), and fluorescent-labeled hybridization probes (Light Cycler), have been commonly used for real-time PCR. Among those chemistries, SYBR Green is the most economical choice. We have used real-time PCR and SYBR Green to examine the expression of a number of leafy spurge genes after growth induction and during normal seasonal growth. Because no reliable endogenous reference genes have been identified in leafy spurge, we performed PCR without an endogenous reference gene and analyzed messenger RNA (mRNA) expression based on the threshold cycle (CT) value of amplification. Excluding an endogenous reference gene from that data analysis was rather straightforward and reliable if RNA was properly prepared and quantified. Given that genomic tools, such as expressed sequence tags (ESTs), and their expression profiles are lacking for most weedy species, avoiding the use of endogenous reference genes in real-time PCR simplifies the optimization process and reduces the cost tremendously. However, we found that using a passive reference dye (ROX) to normalize non-PCR–related fluctuations in fluorescent signal is desirable.

In this review, we examine current techniques and recent advances directed toward understanding cellular mechanisms involved in controlling dormancy in vegetative propagules. Vegetative propagules (including stems, rhizomes, tubers, bulbs, stolons, creeping roots, etc.) contain axillary and adventitious buds capable of producing new stems/branches under permissive environments. Axillary and adventitious buds are distinct in that axillary buds are formed in the axil of leaves and are responsible for production of lateral shoots (branches). Adventitious buds refer to buds that arise on the plant at places (stems, roots, or leaves) other than leaf axils. Both axillary and adventitious buds generally undergo periods of dormancy. Dormancy has been described as a temporary suspension of visible growth of any plant structure containing a meristem (Lang et al. 1987). Dormancy can be subdivided into three categories: (1) ecodormancy-arrest is under the control of external environmental factors; (2) paradormancy-arrest is under the control of external physiological factors within the plant; and (3) endodormancy-arrest is under the control of internal physiological factors. One common feature in all of these processes is prevention of growth under conditions where growth should otherwise continue. There is growing evidence that lack of growth is due to blockage of cell division resulting from interactions between the signaling pathways controlling dormancy and those controlling the cell cycle.

Plant model systems have contributed greatly to the dramatic progress in understanding the fundamental aspects of plant biology. Using model weeds will also help facilitate focused funding and research in the weed science community. Criteria for developing model weeds require attention to weedy characteristics that impart economic losses and a wide geographic distribution, attributes that present the potential for political and scientific support. Expressed sequence tag (EST) databases for model weeds are the most practical approach to identifying new genes and obtaining data on the gene expression underlying weedy characteristics. Weeds such as Canada thistle, eastern black nightshade, johnsongrass, jointed goatgrass, leafy spurge, waterhemp, and weedy rice are proposed as model systems.

Recommended rates of glyphosate for noncultivated areas destroy the aboveground shoots of the perennial plant leafy spurge. However, such applications cause little or no damage to underground adventitious buds (UABs), and thus the plant readily regenerates vegetatively. High concentrations of glyphosate, applied under controlled environmental conditions, have been shown to cause sublethal effects in UABs of leafy spurge that produce stunted and bushy phenotypes in subsequent generations of shoots. We treated leafy spurge plants in the field with glyphosate (0, 1.1, 3.4, or 6.7 kg ai ha−1) to determine its effects on vegetative growth from UABs and on molecular processes. The number of shoots derived from UABs of glyphosate-treated plants was significantly increased compared to controls in subsequent years after application, and new shoots displayed various phenotypical changes, such as stunted and bushy phenotypes. Quantifying the abundance of a selected set of transcripts in UABs of nontreated vs. treated plants (0 vs. 6.7 kg ha−1) indicated that glyphosate impacted molecular processes involved in biosynthesis or signaling of tryptophan or auxin (ARF4, CYP79B2, PIN3, TAA1, TRP6, YUC4), gibberellic acid (GA1/CPS1, GA2/KS), ethylene (ACO1, ACS10), cytokinins (AHP1, AK2, CKX1), and the cell cycle (CDC2A, CDC2B, CYCD3;1). Glyphosate-induced effects on vegetative growth and transcript abundance were persistent for at least 2 yr after treatment. Determining the molecular mechanisms associated with vegetative reproduction in leafy spurge following foliar glyphosate-treatment could identify limiting factors or new targets for manipulation of plant growth and development in perennial weeds.

Long-term control of leafy spurge with glyphosate requires multiple applications because the plant reproduces vegetatively from abundant underground adventitious buds, referred to as crown and root buds. Determining the molecular mechanisms involved in controlling vegetative reproduction in leafy spurge following foliar glyphosate treatment could identify limiting factors or new targets for manipulation of plant growth and development in invasive perennial species. Thus, we treated leafy spurge plants with 0 or 2.24 kg ai ha−1 glyphosate to determine its impact on selected molecular processes in crown buds derived from intact plants and plants decapitated at the soil surface 7 d after glyphosate treatment. New shoot growth from crown buds of foliar glyphosate-treated plants was significantly reduced compared with controls after growth-inducing decapitation, and had a stunted or bushy phenotype. Quantification of a selected set of transcripts involved in hormone biosynthesis and signaling pathways indicated that glyphosate had the most significant impact on abundance of ENT-COPALYL DIPHOSPHATE SYNTHETASE 1, which is involved in a committed step for gibberellin biosynthesis, and auxin transporters including PINs, PIN-LIKES, and ABC TRANSPORTERS. Foliar glyphosate treatment also reduced the abundance of transcripts involved in cell cycle processes, which would be consistent with altered growth patterns observed in this study. Overall, these results suggest that interplay among phytohormones such as auxin, ethylene, and gibberellins affect vegetative growth patterns from crown buds of leafy spurge in response to foliar glyphosate treatment.

Signals from both leaves and apical or axillary meristems of leafy spurge are known to inhibit root bud growth. To test the hypothesis that carbohydrates and growth regulators affect root bud growth, decapitated leafy spurge plants were hydroponically treated with glucose, sucrose, gibberellic acid (GA), abscisic acid (ABA), 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA), and a GA biosynthesis inhibitor, paclobutrazol. Both glucose and sucrose caused suppression of root bud growth at concentrations of 30 mM. The inhibitory effect of sucrose was counteracted by GA at 15 μM. In contrast, BA, ABA, NAA, and paclobutrazol inhibited root bud growth at concentrations as low as 1, 2, 1, and 16 μM, respectively. Sugar and starch levels were also determined in root buds at various times after decapitation. Buds of intact plants contained the highest level of sucrose compared with buds harvested 1, 3, and 5 d after decapitation. To determine how seasonal changes affect root bud dormancy, growth from root buds of field-grown plants was monitored for several years. Root buds of field-grown leafy spurge had the highest level of innate dormancy from October to November, which persisted until a prolonged period of freezing occurred in November or early December. Our data support the hypothesis that carbohydrates may be involved in regulating dormancy status in root buds of leafy spurge.

We have isolated both a genomic and near full length cDNA clone for a D-class cyclin gene from the perennial weed leafy spurge. Sequence analysis indicates that this gene has the highest similarity to CYCLIN D3-2 of Arabidopsis. This gene is preferentially expressed in growing shoot apices and is up-regulated in adventitious buds on resumption of growth following loss of correlative inhibition (apical dominance). CYCLIN D3-2 is also induced in nongrowing adventitious buds of plants treated with gibberellic acid or after removal of leaves—treatments known to up-regulate expression of G1 to S phase transition–specific genes such as HISTONE H3 in adventitious buds. CYCLIN D3-2 was not induced on removal of the apical and axillary buds. Expression of CYCLIN D3-2 is down-regulated in adventitious crown buds during initiation of ecodormancy in early winter. Sequence comparisons of CYCLIN D3-2 with its putative orthologue from Arabidopsis identified several conserved motifs in the promoter region and a conserved region capable of forming a stable hairpin loop in the 5′ untranslated region. Conservation of these noncoding sequences across species strongly suggests they have a regulatory function.

Dormancy is a state of shifted physiological activities with cessation of growth. It occurs in seeds and vegetative propagules and enables plants to survive in adverse growing conditions. Traditional studies on dormancy-related problems have mostly focused on hormone changes along with environmental factors that have achieved great insight on these processes at the physiological level. The molecular nature and cellular basis of signals that carry out the processes of dormancy or dormancy breaking are largely unknown. Recent advances in plant genetics and genomics have provided assorted ways to investigate questions concerning dormancy. Various approaches such as developing genetic maps with DNA-based markers, e.g., amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP), analyzing mutant lines, conducting quantitative trait loci (QTL) analysis, two-dimensional polyacrylamide gel electrophoresis, differential display, microarray, have been performed to resolve different issues related to dormancy. The phenotypic variation in dormant seeds or buds is continuous instead of discrete, and thus QTL analysis is desirable to identify the association between genetically determined phenotypes and specific genetic markers (RFLPs). Some aspects of QTL will be introduced. DNA microarray is a recently developed technology that is used to detect and quantitate large numbers of differences in gene expression simultaneously. We have used the DNA microarray technology to study underground bud dormancy and growth in leafy spurge (Euphorbia esula L.). The principle and versatility of DNA microarray will be introduced, and the strategy for applying this technology will be discussed.

Baseline information on inducing germination of dormant leafy spurge seeds with growth regulators and chemicals is lacking. This study was conducted to survey the effect of various substances on germination of leafy spurge seeds. The nontreated control seeds in this population were nearly fully imbibed in 3 h and displayed approximately 35% germination in 21 d under the normal alternating temperature of 20/30 C (16/8 h). Gibberellic acid (GA3, 10 mM) induced 65% germination at constant temperatures of 20 and 30 C. The alternating temperature increased the effectiveness of 10 mM GA3 with 94% germination, a twofold increase over the control. Nontreated seeds did not germinate at the constant temperatures, suggesting that alternating temperature acts via a GA-independent pathway. Kinetin at 0.1 to 1 mM was no more effective than the control, but a saturated solution of kinetin induced 73% germination. Ethephon at 0.01 to 1 mM induced 58 to 66% germination, although there was little response to different concentrations. Ethylene gas at 1 ppm stimulated germination to 77%, a 1.8-fold increase over the control. Germination of seeds incubated continuously in 1 and 10 mM nitrate displayed 35 and 40% germination, respectively. Seeds pulsed for 24 h with 100 mM nitrate displayed 58% germination after 21 d. Potassium phosphate–citrate buffer (pH 3.4) and its individual components induced 60 to 70% germination. Fluridone (10 and 100 µM), 1-naphthaleneacetic acid (NAA, 0.1 to 10 mM), and ethanol (0.2 to 15%) had no effect on germination, but subsequent elongation in the presence of NAA was inhibited because of swelling of the radicle. This research reveals that GA3 is the most effective growth regulator for germination of dormant leafy spurge seeds, and its effect is independent of temperature.

Genomics programs in the weed science community have not developed as rapidly as that of other crop, horticultural, forestry, and model plant systems. Development of genomic resources for selected model weeds are expected to enhance our understanding of weed biology, just as they have in other plant systems. In this report, we describe the development, characteristics, and information gained from an expressed sequence tag (EST) database for the perennial weed leafy spurge. ESTs were obtained using a normalized cDNA library prepared from a comprehensive collection of tissues. During the EST characterization process, redundancy was minimized by periodic subtractions of the normalized cDNA library. A sequencing success rate of 88% yielded 45,314 ESTs with an average read length of 671 nucleotides. Using bioinformatic analysis, the leafy spurge EST database was assembled into 23,472 unique sequences representing 19,015 unigenes (10,293 clusters and 8,722 singletons). Blast similarity searches to the GenBank nonredundant protein database identified 18,186 total matches, of which 14,205 were nonredundant. These data indicate that 77.4% of the 23,472 unique sequences and 74.7% of the 19,015 unigenes are similar to other known proteins. Further bioinformatics analysis indicated that 2,950, or 15.5%, of the unigenes have previously not been identified suggesting that some may be novel to leafy spurge. Functional classifications assigned to leafy spurge unique sequences using Munich Information Center for Protein or Gene Ontology were proportional to functional classifications for genes of arabidopsis, with the exception of unclassified or unknowns and transposable elements which were significantly reduced in leafy spurge. Although these EST resources have been developed for the purpose of constructing high-density leafy spurge microarrays, they are already providing valuable information related to sugar metabolism, cell cycle regulation, dormancy, terpenoid secondary metabolism, and flowering.

We developed two leafy spurge bacterial artificial chromosome (BAC) libraries that together represent approximately 5× coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the Arizona Genomics Institute. These libraries were used to clone full-length genomic copies of an AP2/ERF transcription factor of the A4 subfamily of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEINS (DREB) known to be differentially expressed in crown buds of leafy spurge during endodormancy, a DORMANCY ASSOCIATED MADS-BOX (DAM) gene, and several FLOWERING LOCUS T (FT) genes. Sequencing of these BAC clones revealed the presence of multiple FT genes in leafy spurge. Sequencing also provided evidence that two different DAM transcripts expressed in crown buds of leafy spurge during endo- and eco-dormancy result from alternate splicing of a single DAM gene. Sequence data from the FT promoters was used to identify several conserved elements previously recognized in Arabidopsis, as well as potential novel transcription factor binding sites that may regulate FT. These leafy spurge BAC libraries represent a new genomics-based tool that complements existing genomics resources for the study of plant growth and development in this model perennial weed. Furthermore, phylogenetic footprinting using genes identified with this resource demonstrate the usefulness of studying weedy species to further our general knowledge of agriculturally important genes.

Bud dormancy is the primary mechanism by which the many perennial weeds escape herbicidal and mechanical control. We developed a 2,654-element Euphorbiaceae cDNA microarray using 1,886 sequenced cDNAs from the model perennial weed leafy spurge, 384 cDNAs from cassava, and 384 control genes from other plant, animal, and bacterial species. This array was used to follow changes in gene expression in root buds of leafy spurge following loss of paradormancy. The differential expression of several genes previously identified as being induced following loss of paradormancy was confirmed by microarray analysis. In addition, genes encoding an asparagine synthase, a phosphate-inducible protein, and a curculin-like (mannose-binding) lectin family protein were found to be rapidly up-regulated upon loss of paradormancy. Several genes involved in flavonoid biosynthesis were found to be rapidly down-regulated upon loss of paradormancy. The potential impact of flavonoid biosynthesis on auxin transport in response to bud growth is discussed.

Leafy spurge seeds are responsive to alternating temperature rather than constant temperature for germination. Transcriptome changes of dry leafy spurge seeds and seeds imbibed for 1 and 3 d at 20 C constant (C) and 20 : 30 C alternating (A) temperature were determined by microarray analysis to examine temperature responsiveness. Principal component analysis revealed differences in the transcriptome of imbibed seeds based on the temperature regime. Computational methods in bioinformatics parsed the data into overrepresented AraCyc pathways and gene regulation subnetworks providing biological context to temperature responses. After 1 d of imbibition, the degradation of starch and sucrose leading to anaerobic respiration were common pathways at both temperature regimes. Several overrepresented pathways unique to 1 d A were associated with generation of energy, reducing power, and carbon substrates; several of these pathways remained overrepresented and up-regulated at 3 d A. At 1 d C, pathways for the phytohormones jasmonic acid and brassinosteroids were uniquely overrepresented. There was little similarity in overrepresented pathways at 1 d C between leafy spurge and arabidopsis seeds, indicating species-specific effects upon imbibition of dry seeds. Overrepresented gene subnetworks at 1 d and 3 d at both temperature regimes related to signaling processes and stress responses. A major overrepresented subnetwork unique to 1 d C related to photomorphogenesis via the E3 ubiquitin ligase COP1. At 1 d A, major overrepresented subnetworks involved circadian rhythm via LHY and TOC1 proteins and expression of stress-related genes such as DREB1A, which is subject to circadian regulation. Collectively, substantial differences were observed in the transcriptome of leafy spurge seeds imbibed under conditions that affect the capacity to germinate.