Project description:Leafy spurge (Euphorbia esula) is an herbaceous perennial weed that produces vegetatively from an abundance of underground adventitious buds. The objectives of this study were to determine how mimicking natural seasonal conditions (photoperiod and temperature) under controlled environmental conditions affect dormancy and flowering competence; to determine molecular mechanisms associated with well-defined phases of seasonal dormancy transitions based on transcript profiles obtained by microarray analysis; and to link mechanisms regulating induction and release of endodormancy and flowering competence. Reduction in temperature (27 to 10°C) and photoperiod (16 to 8 h) over a three-month period induced a para- to endo-dormant transition in crown buds. An additional eleven weeks of prolonged cold (5-7°C) and short-photoperiod treatment resulted in accelerated shoot growth from crown buds, and 99% floral competence when plants were returned to growth promoting conditions. Exposure of paradormant plants to short-photoperiod and prolonged cold treatment alone had minimal affect growth potential or on flowering (~1%); whereas endodormant crown buds without prolonged cold treatment, had delayed shoot growth and approximately 2% flowering when returned to growth promoting conditions. Transcriptome analyses revealed that 373 and 260 genes were differentially expressed (p<0.005) during para- to endo-dormant and endo- to eco-dormant transitions, respectively. Transcripts from flower competent vs. non-flower competent crown buds identified 607 differentially expressed genes, and genes involved in cell cycle and DNA processing, oxidative stress, flower regulation, and proteolysis were over-represented. Further, sub-network analysis identified expression targets and binding partners associated with circadian clock, dehydration/cold signaling, phosphorylation cascades, and response to abscisic acid, ethylene, gibberellic acid, and jasmonic acid, suggesting these central regulators affect well-defined phases of dormancy. Potential genetic pathways associated with these dormancy transitions and flowering were used to develop a proposed conceptual model.

Project description:Leafy spurge (Euphorbia esula) is an herbaceous perennial weed that produces vegetatively from an abundance of underground adventitious buds. The objectives of this study were to determine how mimicking natural seasonal conditions (photoperiod and temperature) under controlled environmental conditions affect dormancy and flowering competence; to determine molecular mechanisms associated with well-defined phases of seasonal dormancy transitions based on transcript profiles obtained by microarray analysis; and to link mechanisms regulating induction and release of endodormancy and flowering competence. Reduction in temperature (27 to 10Â°C) and photoperiod (16 to 8 h) over a three-month period induced a para- to endo-dormant transition in crown buds. An additional eleven weeks of prolonged cold (5-7Â°C) and short-photoperiod treatment resulted in accelerated shoot growth from crown buds, and 99% floral competence when plants were returned to growth promoting conditions. Exposure of paradormant plants to short-photoperiod and prolonged cold treatment alone had minimal affect growth potential or on flowering (~1%); whereas endodormant crown buds without prolonged cold treatment, had delayed shoot growth and approximately 2% flowering when returned to growth promoting conditions. Transcriptome analyses revealed that 373 and 260 genes were differentially expressed (p<0.005) during para- to endo-dormant and endo- to eco-dormant transitions, respectively. Transcripts from flower competent vs. non-flower competent crown buds identified 607 differentially expressed genes, and genes involved in cell cycle and DNA processing, oxidative stress, flower regulation, and proteolysis were over-represented. Further, sub-network analysis identified expression targets and binding partners associated with circadian clock, dehydration/cold signaling, phosphorylation cascades, and response to abscisic acid, ethylene, gibberellic acid, and jasmonic acid, suggesting these central regulators affect well-defined phases of dormancy. Potential genetic pathways associated with these dormancy transitions and flowering were used to develop a proposed conceptual model. Leafy spurge is an herbaceous perennial weed that undergoes vegetative reproduction from an abundance of underground adventitious buds which exhibit phases of para-, endo-, and eco-dormancy (defined by Lang et al. 1987) during summer, fall, and winter, respectively (Anderson et al. 2005). In this study a population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype 1984-ND001. Paradormant crown buds from three-month old greenhouse plants were collected after one week of acclimation under growth chamber conditions. Induction of endodormancy in crown buds was accomplished by subjecting greenhouse grown and growth chamber acclimated plants to a ramp down (RD) treatment consisting of a reduction in temperature of 1.42Â°C week-1 (27Â°C â 10Â°C) and a decreasing photoperiod of 40 min week-1 (16h â 8h light) for 12 weeks as previously established by Foley et al. (2009). These conditions mimic the average seasonal environmental conditions experienced in Fargo, ND (46Â°54â² N, 96Â°48â² W) during the transition from para- to endo-dormancy (Anderson et al. 2005). To induce a transition of crown buds from endo- to eco-dormancy, plants subjected to the RD treatment were given prolonged cold treatment for 11 weeks at 5-7Â°C, under constant 8 h:16 h day:night cycle; light fluencies were approximately 250 Î¼mol m-2 s-1. To compare the effects of the prolonged cold with or without RD treatment on dormancy status and flowering competence in crown buds, greenhouse-grown and growth chamber acclimated plants were subjected directly to an extended cold treatment for 11 weeks at 5-7Â°C, as previously described, without a RD treatment. For microarray hybridizations, labeled cDNAs were prepared from 30 Âµg of total RNA using the Alexa Fluor cDNA labeling kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocols. Labeled cDNAs were hybridized to a custom made 23K element microarray that contained 19,808 unigenes from a leafy spurge EST database (Anderson et al. 2007) and an additional 4,129 unigenes from a cassava EST database (Lokko et al. 2007). Comparison of gene expression between samples was accomplished using a rolling circle dye swap hybridization scheme (Churchill 2002) to provide every biological replicate with technical replicates. In our microarray analyses experiment, each of the four biological replicates included four technical replicates using two different dyes, resulting in a total of 16 technical replicates for each treatment. Microarray hybridization was visualized using a GenePix 4000B scanner and probe intensities and background were quantified using GenePix 6.0 software (Molecular Devices, Sunnyvale, California, USA). A quality control value of â1â was assigned to all probes that had intensity values greater than 2 times the standard deviation over average of the negative control and empty probe intensities (after deletion of 1% of the most intense negative/empty probe values). Hybridization intensities were log2 transformed, and arrays were centered and normalized against each other.

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