Transcriptome analysis of time couse of leafy spurge infection with Xam
ABSTRACT: The goal of this study was to identify signaling processes associated with infection and recovery of leafy spurge inoculated with Xanthomonas axonopodis pv. manihotis (Xam). Two dye-swap technical replicates for each of 3 biological replicates for each treatment were analyzed. A time course analysis of Xam infected and mock inoculated leafy spurge was performed. Time points were 1, 7 and 21 days after inoculation (or mock inoculation). Thus, 36 labeled samples were hybridized in a 2 dye rolling circle hybridization scheme to 18 arrays.
Project description:Transcriptome changes were investigated for Euphorbia esula (leafy spurge) seeds with a focus on the effect of constant and diurnal fluctuating temperature on dormancy and germination. Leafy spurge seeds do not germinate when incubated for 21 days at 20°C constant temperatures, but nearly 30% germinate after 21 days under fluctuating temperatures 20:30°C (16:8 h). Incubation at 20°C for 21 followed by 20:30°C resulted in approximately 63% germination in about 10 days. A cDNA microarray representing approximately 22,000 unique sequences was used to profile transcriptome changes. Labeled cDNA was prepared from total RNA using the Alexa Fluor cDNA labeling kit (Invitrogen, Carlsbad, CA) according to manufacture's protocols. Labeled cDNAs were hybridized to a custom made 23 K element microarray that contained 19,808 unigenes from the leafy spurge EST database and an additional 4,129 unigenes from a cassava EST database. A rolling circle dye swap hybridization scheme was used to compare gene expression between samples. There were three biological and two technical replications for each treatment. Microarray hybridization was visualized using a GenPix 4000B scanner (Axon Instruments/Molecular Devices Corp., Sunnyvale, CA) and spot intensities and background was quantified using GenPix Pro software. Hybridization intensities were log2 transformed, and arrays were centered and normalized against each other.
Project description:This study investigated changes in the transcriptome of outdoor grown leafy spurge crown buds as they progress from paradormancy in August and September into endo dormancy in October through to ecodormancy in November and December. Keywords: Dormancy leafy spurge adventitious-buds A series of balanced dyeswap rolling circle hybiridizations were used with each year representing s seperate circle and with direction of the circles reversing on alternate years. Note, the Aug-Sep 04 hybridization failed and is missing from the dataset
Project description:This submission contains the RNAseq data from a study of leafy spurge crown buds transitioning through a seasonal dormancy time course where buds transitioned from paradoprmancy to endodormancy and then to ecodormancy. The sequences in this study were mapped to an assembled transcriptome built from sequences from this study along with sequences from : 1) A study of leafy spurge crown buds through a time course for paradormancy release induced by excision of the aerial portion of the shoot (Series GSE71317). 2) A study identical to this study but where leafy spurge plants were first treated with glyphosate (Series GSE71406). 3) A previously submitted study of leafy spurge shoots following treatment with glyphosate was also used to assemble the transcriptome (Series GSE56509). This crown buds transitioning through a seasonal dormancy time course study has 4 biological replicates collected at each of the three dormancy states (paradormant, endodormant, and ecodormant).
Project description:This submission contains the RNAseq data from one (just the study on paradormancy release) of several studies used to assemble the leafy spurge tranacriptome. These assembled transcrptome was built from 1) A study of leafy spurge crown buds through a time course for paradormancy release induced by excision of the aerial portion of the shoot (this study). 2) A study of leafy spurge crown buds through a seasonal dormancy time course where buds transitioned from paradoprmancy to endodormancy and then to ecodormancy (Series GSE71321). 3) A study identical to #2 above but where leafy spurge plants were first treated with glyphosate (Series GSE71406). 4) A previously submitted study (Series GSE56509) of leafy spurge shoots following treament with glyphosate was also used to assemble the transcriptome. The paradormancy study (#1) contained 4 time points (0hr, 6h, 1d, 3d) with 3 biological replicates at 0hr and 4 biological replicates at each of the other time points.
Project description:This submission contains the RNAseq data from a study of leafy spurge crown buds transitioning through a seasonal dormancy time course following glyphosate treatments where buds transitioned from paradoprmancy to endodormancy and then to ecodormancy. The sequences in this study were mapped to an assembled transcriptome built from sequences from this study along with sequences from : 1) A study of leafy spurge crown buds through a time course for paradormancy release induced by excision of the aerial portion of the shoot (Series GSE71317). 2) A study identical to this study but where leafy spurge plants were not treated with glyphosate (Series GSE71321). 3) A previously submitted study of leafy spurge shoots following treament with glyphosate was also used to assemble the transcriptome (Series GSE56509). This crown buds transitioning through a seasonal dormancy time course following a glyphosate treatment study has 4 biological replicates collected at each of the three dormancy states (paradormant, endodormant, and ecodormant).
Project description:New shoot growth from underground adventitious buds of leafy spurge is critical for survival of this invasive perennial weed after episodes of severe abiotic stress. Because global climate change is expected to increase abiotic stress, such as dehydration, objectives of this study include examining the impact that dehydration stress has on molecular mechanisms associated with vegetative reproduction. Greenhouse plants were exposed to mild- (3-day), intermediate- (7-day), severe- (14-day) and extended- (21-day) dehydration treatments, prior to decapitation of aerial tissue and rehydration of soil to induce new vegetative shoot growth. Compared to well-watered control plants, mild-dehydration accelerated new vegetative shoot growth but intermediate- and severe-dehydration treatments both delayed and reduced shoot growth, and 21-day dehydration treatment inhibited initiation of new vegetative shoots and was considered a lethal treatment. Overall, transcriptome profiles revealed that 2109 genes were differentially-expressed (P<0.05) in crown buds in response to the various dehydration treatments. Sub-network enrichment analyses identified central hubs of over-represented genes involved in processes such as hormone responses and signaling (e.g., ABA, auxin, ethylene, GA, and JA), response to abiotic stress (DREB1A/2A) and light (PIF3), phosphorylation (CLV1, MPK3/4/6, SOS2), gene silencing (miRNA156/172a), circadian regulation (CRY2, LHY, PHYA/B), and flowering (AGL8/20, AP2, FLC). Further, results from this and previous studies highlight HY5, MAF3, MYB-like/RVE1 and RD22 as molecular markers for endodormancy in crown buds of leafy spurge. Early response to dehydration also highlighted involvement of upstream ethylene and jasmonate signaling, whereas longer-term dehydration impacted ABA signaling. The identification of conserved ABRE- and MYC-consensus, cis-acting elements in the promoter of a leafy spurge gene similar to Arabidopsis MYB-like/RVE1 (AT5G17300) implicates a potential role for ABA signaling in its dehydration-induced expression. Response of these molecular mechanisms to dehydration-stress provides insights on the ability of invasive perennial weeds to adapt and survive under harsh environments, which provide new insights for addressing future management practices. Changes in transcript abundance for underground adventitious buds of leafy spurge which were exposed various levels of dehydration stress (Day-3, -7, -14, -16, -21) are analysed relative to controls (Day-0).
Project description:Glyphosate is known to inhibit 5-enolpyruvylshikimate-3-phosphate synthase of the chorismate biosynthetic pathway, and chorismate is a precursor to aromatic amino acids, auxin, and many other secondary products. Although the perennial weed leafy spurge (Euphorbia esula L.) is considered glyphosate tolerant, glyphosate is often used as part of an integrated pest management program in non-cultivated ecosystems of North America. Part of its tolerance is attributed to escape through an abundance of underground adventitious buds (UABs). Sub-lethal concentrations of foliar applied glyphosate leads to new shoot growth from UABs that have a stunted and/or bushy phenotype after growth-inducing decapitation. To gain insights into glyphosate’s impact on molecular mechanisms associated with the stunted and bushy phenotype, we obtained global transcriptome abundance using RNAseq from a subsequent generation of aerial shoots derived from crown buds of glyphosate-treated and -untreated leafy spurge. We further correlated transcript abundance to accumulation of shikimate and phytohormones from the same samples to elucidate interactions. Abundance of shikimate was similar in subsequent generations of aerial shoots generated from crown buds of treated and untreated plants and is likely not a direct factor leading to the stunted and bushy phenotype. However, the results do suggest that transcripts involved in auxin transport and signaling and crosstalk with other phytohormones likely play a role in the bushy phenotype. The results of this study provide some insights for identifying new targets for manipulation of plant growth and development. Transcriptome and metabolite profiling are obtained for aerial tissues derived from crown buds of foliar glyphosate-treated and control (2.24 or 0 kg/ha active ingredient glyphosate + 0.25% v/v surfactant) leafy spurge plants. Each experiment included 4 biological replicates.
Project description:Persistence of the invasive perennial weed leafy spurge is mainly attributed to seasonal production of underground adventitious buds (UABs), which undergo well-defined phases of dormancy (para-, endo- and eco-dormancy). These well-defined phases of dormancy also allow UABs of leafy spurge to survive extreme seasonal variations, including dehydration-stress. Consequently, objectives of this study include understanding the effects of dehydration-stress on vegetative reproduction, flowering competence, and transcript profiles at different phases of bud dormancy. The vegetative growth potential of UABs was monitored by removing the aerial portion of dehydration-stressed plants and re-watering the root system. Further, microarray analysis was used to follow transcriptome profiles to identify critical defense- and signaling-pathways at different phases of dormancy in UABs. Surprisingly, only 3 days of dehydration-stress is required to break the endodormant phase in UABs. In leafy spurge, vernalization of endodormant UABs has previously been shown to induce flower competence, while breaking of endodormancy via dehydration-stress did not induce floral induction. Thus, these two environmental treatments open a unique approach to independently dissect molecular mechanisms involved in endodormancy maintenance and floral competence. Bioinformatics analysis of transcriptome data helped to identify models and overlapping pathways. During endodormancy break, LEC1, PHOTOSYSTEM I RC, and brassinosteroids were identified as ooverlapping hubs of up-regulated genes, and DREB1A, CBF2, GPA1, MYC2, BHLH, BZIP, and flavonoids were identified as overlapping hubs of down-regulated genes. Additionally, key genes involved in metabolic activity, chromatin modification, and cross-talk between growth regulators were identified as playing a role during endodormancy maintenance. Plant material, controlled environmental treatments, and vegetative growth: Three-month-old paradormant leafy spurge plants grown in a greenhouse were acclimated for one week, under growth chamber conditions, prior to being subjected to a ramp-down in temperature (27 °C → 10 °C) and photoperiod (16 h → 8 h light) for 12 weeks to induce endodormancy, as previously established (Doğramacı et al. 2010; Foley et al. 2009). Endodormancy status was confirmed by comparing vegetative growth rates of plants with and without a ramp-down treatment. To study the effects of dehydration-stress on vegetative growth from UABs, water was withheld from endodormant plants up to 21 days. After dehydration for 0, 1-, 3-, 7-, 14-, and 21-days, the aerial portion of the plants were decapitated at the soil surface and vegetative growth and floral competence of UABs were monitored under growth-conducive conditions by re-watering the root system. Vegetative shoot growth from six plants was recorded weekly for four weeks, and results of four replications were analyzed using SAS 9.2 (SAS Institute, Cary, NC, 2008) software as described by Doğramacı et al. (2010). Microarray analysis: At the end of each treatment, crown buds were collected, and RNA extraction, cDNA synthesis, fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described by Doğramacı et al. (2010). Based on unexpected vegetative growth results obtained from this study, only microarray data obtained from 0-, 1-, and 3-day dehydration-stressed endodormant UABs, which were compared with microarray data for endodormant, and flowering competent ecodormant UABs from our previous study (Doğramacı et al. 2010; GSE19217), were used for bioinformatics analyses. However, to merge the datasets from these two separate experiments, T-tests were first performed on microarray data obtained from endodormant UABs from each experiment, and differentially-expressed genes (p<0.005) were removed from the dataset (~5% of the spots). After removing the outliers, the two endodormant transcriptome data sets were grouped together and used as the baseline control for further ANOVA, T-tests and other analyses. Array normalization, statistical analyses and clustering of the dataset and Venn diagram generation were done using GeneMaths XT 5.1 software as previously described by Doğramacı et al. (2010).
Project description:Leafy spurge is a model for studying well-defined phases of dormancy in underground adventitious buds (UABs) of herbaceous perennial weeds, which is a primary factor allowing many invasive perennial weeds to escape conventional control measures. A 12-week ramp down in both temperature (27°C → 10°C) and photoperiod (16 h → 8 h light) is required to induce a transition from para- to endo-dormancy in UABs of leafy spurge. To evaluate the effects of photoperiod and temperature on molecular networks associated with this transition, we compared global transcriptome data-sets obtained from UABs of leafy spurge exposed to a ramp down in both temperature and photoperiod (RDtp) vs. a ramp down in temperature (RDt) alone. Analysis of transcriptome data-sets indicated that numerous genes associated with circadian clock, photoperiodism, flowering, and hormone responses (CCA1, COP1, HY5, MAF3, MAX2) were preferentially expressed during the transition from para- to endo-dormancy. Gene-set enrichment analyses highlighted metabolic pathways associated with ethylene, auxin, flavonoids, and carbohydrate metabolism; whereas, sub-network enrichment analyses identified hubs (CCA1, CO, FRI, mir172A, EINs, DREBs) of gene networks associated with carbohydrate metabolism, circadian clock, flowering, and stress and hormone responses during the transition to endodormancy. These results helped refine existing models for the transition to endodormancy in UABs of leafy spurge, which strengthened the roles of circadian clock associated genes, DREBs, COP1-HY5, carbohydrate metabolism, and involvement of hormones (ABA, ethylene, and strigolactones). Further, we propose that the RDtp treatment ultimately leads to a chain effect, responsive to photoperiod and temperature signaling, to synchronize molecular processes associated with the transition from para- to endo-dormancy. Plant material, environmental treatments, and vegetative growth Leafy spurge plants were propagated from the uniform biotype (1984-ND001) and maintained in a greenhouse as described by Anderson and Davis (2004). Prior to the start of each experiment, plants were acclimated in a Conviron growth chamber (Model PGR15) for one week at 27°C, 16:8 h light:dark photoperiod. Each experiment was replicated four times, and each replicate contained 30 plants. Six plants from each replicate were used to determine vegetative growth rate, and crown buds from the remaining 24 plants were collected for transcriptome studies. All samples were collected between 11:00 and 13:00 a.m. to avoid diurnal variation. We previously established conditions for induction and release of dormancy phases under controlled environments (Doğramacı et al. 2010; Foley et al. 2009). To induce endodormancy, paradormant plants were subjected to a ramp-down in temperature (27°C → 10°C) and photoperiod (16 h → 8 h light), i.e., RDtp, for 12 weeks (Fig. 1, Exp-1). To distinguish the individual effects of temperature and photoperiod on molecular networks involved in endodormancy induction (see Fig. 1, Exp-2), we compared three-month old paradormant plants subjected to a ramp-down in temperature (27°C → 10°C) under constant photoperiod (16 h light) for 12 weeks (i.e., RDt) to RDtp plants. Additionally, a set of paradormant plants were kept under constant temperature and light (27°C, 16 h light) as a control. At the end of each treatment, the aerial portion of the plant was decapitated to determine the dormancy status of the crown buds by their vegetative growth rate in the greenhouse, as described by Foley et al. (2009). New vegetative shoot growth was recorded weekly; results were analyzed using the generalized linear mixed model (PROC GLIMMIX) procedure of SAS 9.2, and 95% confidence intervals for treatment by week means were generated. Transcriptome analyses At the end of each treatment, crown buds were collected from intact plants to study transcriptome profiles and were stored at -80°C. RNA extraction, cDNA synthesis and fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described (Doğramacı et al. 2010). Transcriptome data obtained from Exp-1 and -2 (Fig. 1) was used for various bioinformatics analysis. GeneMaths XT 2.1 software was used to normalize the arrays, to conduct statistical analyses, and to generate Venn diagrams as previously described by Doğramacı et al. (2010). Expression data for both Exp-1 and -2 are deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as GEO dataset query GSE19217 and GSE??, respectively. The entire data set was further analyzed using Ariadne Pathway Studio 7 Software-Resnet Plant Version 2.1 (Ariadne Genomics Inc., Rockville, MD, USA) to obtain Gene Set Enrichment Analysis (GSEA) and Sub Network Enrichment Analysis (SNEA). GSEA is used to determine if predefined sets of genes are over-represented (P<0.05) between treatments; focusing on gene sets based on AraCyc metabolic pathways http://pmn.plantcyc.org/ARA/class-instances?object=Pathways, and gene ontology (GO) (http://www.geneontology.org/). SNEA algorithm was used to identify the networks by highlighting over-represented ontologies based on published gene regulation hierarchies, protein:protein interactions, or protein modification targets. Furthermore, these networks were visualized using the Union Selected Pathway function of the software for expression targets, binding partners, protein modification targets, and also custom advanced function to generate sub-networks as neighbors of proteins based on expression, regulation, molecular transport, protein modification, promoter binding, molecular synthesis, chemical reaction, and direct regulation.
Project description:This study is a time course of growth induction (0, 12, 24, 48, and 72 hrs) following the breaking of paradormancy in underground buds of the perennial weed leafy spurge (Euphorbia esula). Keywords: Time course, growth induction, apical dominance, paradormancy, root buds, leafy spurge Overall design: Two independently isolated samples for each time point was compared to a single control sample (also independently isolated) collected from paradormant underground adeventitious buds. Dye swaps were done for each time point (not each biological replicate).