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Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula).

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis “general purpose” traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the CT values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCT method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species.

Dehydration and vernalization treatments identify overlapping molecular networks impacting endodormancy maintenance in leafy spurge crown buds

Leafy spurge (Euphorbia esula L.) is a herbaceous perennial weed that reproduces vegetatively from an abundance of underground adventitious buds (UABs), which undergo well-defined phases of seasonal dormancy (para-, endo-, and ecodormancy). In this study, the effects of dehydration stress on vegetative growth and flowering potential from endodormant UABs of leafy spurge was monitored. Further, microarray analysis was used to identify critical signaling pathways of transcriptome profiles associated with endodormancy maintenance in UABs. Surprisingly, only 3-day of dehydration stress is required to break the endodormant phase in UABs; however, the dehydration-stress treatment did not induce flowering. Previous studies have shown that prolonged cold treatment of UABs breaks endodormancy and induces a vernalization response leading to flowering. Thus, in this study, comparing transcriptome data from UABs exposed to short-term dehydration and vernalization provided a unique approach to identify overlapping molecular mechanisms involved in endodormancy maintenance and floral competence. Analysis of transcriptome data associated with breaking endodormancy by both environmental treatments identified LEC1, PHOTOSYSTEM I RC, and brassinosteroids as common central hubs of upregulated genes, while DREB1A, CBF2, GPA1, MYC2, bHLH, BZIP, and flavonoids were identified as common central hubs of downregulated genes. The majority of over-represented gene sets common to breaking endodormancy by dehydration stress and vernalization were downregulated and included pathways involved in hormone signaling, chromatin modification, and circadian rhythm. Additionally, the over-represented gene sets highlighted pathways involved in starch and sugar degradation and biogenesis of carbon skeletons, suggesting a high metabolic activity is necessary during the endodormant phase. The data presented in this study helped to refine our previous model for dormancy regulation.

Glyphosate’s impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching

BackgroundLeafy spurge (Euphorbia esula) is a perennial weed that is considered glyphosate tolerant, which is partially attributed to escape through establishment of new vegetative shoots from an abundance of underground adventitious buds. Leafy spurge plants treated with sub-lethal concentrations of foliar-applied glyphosate produce new vegetative shoots with reduced main stem elongation and increased branching. Processes associated with the glyphosate-induced phenotype were determined by RNAseq using aerial shoots derived from crown buds of glyphosate-treated and -untreated plants. Comparison between transcript abundance and accumulation of shikimate or phytohormones (abscisic acid, auxin, cytokinins, and gibberellins) from these same samples was also done to reveal correlations.ResultsTranscriptome assembly and analyses confirmed differential abundance among 12,918 transcripts (FDR ≤ 0.05) and highlighted numerous processes associated with shoot apical meristem maintenance and stem growth, which is consistent with the increased number of actively growing meristems in response to glyphosate. Foliar applied glyphosate increased shikimate abundance in crown buds prior to decapitation of aboveground shoots, which induces growth from these buds, indicating that 5-enolpyruvylshikimate 3-phosphate (EPSPS) the target site of glyphosate was inhibited. However, abundance of shikimate was similar in a subsequent generation of aerial shoots derived from crown buds of treated and untreated plants, suggesting EPSPS is no longer inhibited or abundance of shikimate initially observed in crown buds dissipated over time. Overall, auxins, gibberellins (precursors and catabolites of bioactive gibberellins), and cytokinins (precursors and bioactive cytokinins) were more abundant in the aboveground shoots derived from glyphosate-treated plants.ConclusionBased on the overall data, we propose that the glyphosate-induced phenotype resulted from complex interactions involving shoot apical meristem maintenance, hormone biosynthesis and signaling (auxin, cytokinins, gibberellins, and strigolactones), cellular transport, and detoxification mechanisms.

The transcriptomes of dormant leafy spurge seeds under alternating temperature are differentially affected by a germination-enhancing pretreatment

Seed dormancy is an important stage in the life cycle of many non-domesticated plants, often characterized by the temporary failure to germinate under conditions that normally favor the process. Pre-treating dormant imbibed seeds at a constant temperate accelerated germination of leafy spurge seeds under alternating temperatures. However, dormant seeds will also germinate without a pre-treatment, albeit at a much slower rate, which gives rise to longer periods of imbibition before germination. Transcriptome analyses on seeds exposed to prolonged imbibition highlighted pathways associated with phenylpropanoid biosynthesis and interacting networks of genes involved in plant defense. In addition to the many pathways associated with phenylpropanoid biosynthesis enriched with down-regulated genes upon germination, there were also numerous pathways enriched with up-regulated genes associated with energy metabolism, such as glycolysis. Transcriptome data further suggest that metabolism and signaling by the plant hormones ethylene, gibberellin, and abscisic acid are involved in the developmental transition from dormancy to germination. More specifically, sub-network enrichment analysis identified ABI3 as a central hub of a sub-network at germination including several down-regulated genes such as DELLA (i.e., RGL2), which represses gibberellin signaling processes required for germination. The 595-fold increase in the expression of ACC oxidase (ACO4) at germination also suggests an important role for ethylene biosynthesis in germinating leafy surge seeds. Furthermore, the 10-578-fold difference in expression of many genes such as HY5 and Histone H3 between two populations at germination, which were treated with and without a constant temperature germination-enhancing pretreatment, revealed disparate impacts on various biosynthetic, growth, signaling, and response processes. Overall, our results indicate a constant temperature pretreatment (20°C for 21d) is not required for germination of leafy spurge seeds at an alternating temperature. However, the presence or absence of the pretreatment does affect the rate of germination and the germination transcriptional programs.

Comparison of phytohormone levels and transcript profiles during seasonal dormancy transitions in underground adventitious buds of leafy spurge

Leafy spurge (Euphorbia esula L.) is an herbaceous perennial weed that maintains its perennial growth habit through generation of underground adventitious buds (UABs) on the crown and lateral roots. These UABs undergo seasonal phases of dormancy under natural conditions, namely para-, endo-, and ecodormancy in summer, fall, and winter, respectively. These dormancy phases can also be induced in growth chambers by manipulating photoperiod and temperature. In this study, UABs induced into the three phases of dormancy under controlled conditions were used to compare changes in phytohormone and transcriptome profiles. Results indicated that relatively high levels of ABA, the ABA metabolite PA, and IAA were found in paradormant buds. When UABs transitioned from para- to endodormancy, ABA and PA levels decreased, whereas IAA levels were maintained. Additionally, transcript profiles associated with regulation of soluble sugars and ethylene activities were also increased during para- to endodormancy transition, which may play some role in maintaining endodormancy status. When crown buds transitioned from endo- to ecodormancy, the ABA metabolites PA and DPA decreased significantly along with the down-regulation of ABA biosynthesis genes, ABA2 and NCED3. IAA levels were also significantly lower in ecodormant buds than that of endodormant buds. We hypothesize that extended cold treatment may trigger physiological stress in endodormant buds, and that these stress-associated signals induced the endo- to ecodormancy transition and growth competence. The up-regulation of NAD/NADH phosphorylation and dephosphorylation pathway, and MAF3-like and GRFs genes, may be considered as markers of growth competency.

Foliar Application of Glyphosate Affects Molecular Mechanisms in Underground Adventitious Buds of Leafy Spurge (Euphorbia esula) and Alters Their Vegetative Growth Patterns

Abstract 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. Nomenclature: Glyphosate; leafy spurge, Euphorbia esula L.

BAC Library Development and Clone Characterization for Dormancy-Responsive DREB4A, DAM, and FT from Leafy Spurge (Euphorbia esula) Identifies Differential Splicing and Conserved Promoter Motifs

Abstract 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. Nomenclature: Arabidopsis, Arabidopsis thaliana L. ARATH; leafy spurge, Euphorbia esula L. EPHES.

Changes in the Transcriptome of Dry Leafy Spurge (Euphorbia esula) Seeds Imbibed at a Constant and Alternating Temperature

Abstract 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. Nomenclature: Mouse-ear cress, Arabidopsis thaliana (L.) Heynh.; leafy spurge, Euphorbia esula L. (EPHES).

Gene Space and Transcriptome Assemblies of Leafy Spurge (Euphorbia esula) Identify Promoter Sequences, Repetitive Elements, High-Quality Markers, and a Full-Length Chloroplast Genome

Abstract 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 proteincoding 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.

Field Application of Glyphosate Induces Molecular Changes Affecting Vegetative Growth Processes in Leafy Spurge (Euphorbia esula)

Abstract 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. Nomenclature: Glyphosate, leafy spurge, Euphorbia esula L.