species: Leafy spurge tissue: crown buds treatment: exposed to ramp down in temperature under constant photoperiod for 12 weeks, then vernalized time: 12 weeks, then vernalized
Biomaterial provider
Munevver Dogramaci
Treatment protocol
We previously established conditions for induction and release of dormancy phases under controlled environments (Doğramacı et al. 2010, 2011; 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. In this study, to distinguish the individual effects of temperature and photoperiod on molecular networks involved in endodormancy induction, 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. At the end of each treatment, crown bud samples were collected between 1100 and 1300 Central Standard times to avoid diurnal variation. Samples were collected after 3, 6, 9, 12 week during RDt treatment. Additionally, a set of paradormant plants were kept under constant temperature and light (27°C, 16 h light) as a control (Para-0 week); we also maintained a set of paradormant plants, as a second control, in the greenhouse and collected samples at the end of 12 weeks of RDt treatment (Para-12 week). This sample represents crown buds collected from plants exposed to ramp down in temperature for 12 weeks under constant light, and then exposed to vernalizing cold treatment (5-7°C for 8 weeks).
Growth protocol
A population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype 1984-ND001 and maintained in a greenhouse as described by Anderson and Davis (2004). Shoot cuttings were grown in No. 1 Ray Leach Cone-tainers (Stuewe and Sons Inc. Corvallis, OR, USA) in the greenhouse for three months prior to application of experimental treatments. The greenhouse was set at 25°C with a 16 h:8 h day:night photoperiod. Average daily light fluencies were approximately 350 μmol m-2 s-1 and 400 W high-pressure sodium lamps were used as to extend the day-length. Each treatment was replicated four times, and thirty plants were used per biological replication. Prior to the start of each experiment, plants were randomly chosen from the greenhouse and acclimated in a growth chamber (PGR15 Model of Conviron, Winnipeg, Canada) for one week at 27°C, 16 h:8 h day:night photoperiod. At the end of each treatment, crown bud samples were collected between 1100 and 1300 Central Standard times to avoid diurnal variation.
Extracted molecule
total RNA
Extraction protocol
At the end of each treatment, crown bud samples were collected, flash froze in liquid N2, and stored at -80°C until RNA extraction. Crown bud samples were ground to a fine powder in liquid N2, and RNA was extracted from approximately one gram of tissue according to the pine tree RNA extraction protocol (Chang et al. 1993). RNA quality and quantity was confirmed by spectrophotometry and agarose gel electrophoresis.
Label
CY5
Label protocol
For microarray hybridizations, labeled cDNAs were prepared from 30 µg 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 this experiment, each of the four biological replicates included two technical replicates.
We previously established conditions for induction and release of dormancy phases under controlled environments (Doğramacı et al. 2010, 2011; 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. In this study, to distinguish the individual effects of temperature and photoperiod on molecular networks involved in endodormancy induction, 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. At the end of each treatment, crown bud samples were collected between 1100 and 1300 Central Standard times to avoid diurnal variation. Samples were collected after 3, 6, 9, 12 week during RDt treatment. Additionally, a set of paradormant plants were kept under constant temperature and light (27°C, 16 h light) as a control (Para-0 week); we also maintained a set of paradormant plants, as a second control, in the greenhouse and collected samples at the end of 12 weeks of RDt treatment (Para-12 week). This sample represents crown buds collected from paradormant crown buds used as secondary controls, to complement 12 week ramp down treatment.
Growth protocol
A population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype 1984-ND001 and maintained in a greenhouse as described by Anderson and Davis (2004). Shoot cuttings were grown in No. 1 Ray Leach Cone-tainers (Stuewe and Sons Inc. Corvallis, OR, USA) in the greenhouse for three months prior to application of experimental treatments. The greenhouse was set at 25°C with a 16 h:8 h day:night photoperiod. Average daily light fluencies were approximately 350 μmol m-2 s-1 and 400 W high-pressure sodium lamps were used as to extend the day-length. Each treatment was replicated four times, and thirty plants were used per biological replication. Prior to the start of each experiment, plants were randomly chosen from the greenhouse and acclimated in a growth chamber (PGR15 Model of Conviron, Winnipeg, Canada) for one week at 27°C, 16 h:8 h day:night photoperiod. At the end of each treatment, crown bud samples were collected between 1100 and 1300 Central Standard times to avoid diurnal variation.
Extracted molecule
total RNA
Extraction protocol
At the end of each treatment, crown bud samples were collected, flash froze in liquid N2, and stored at -80°C until RNA extraction. Crown bud samples were ground to a fine powder in liquid N2, and RNA was extracted from approximately one gram of tissue according to the pine tree RNA extraction protocol (Chang et al. 1993). RNA quality and quantity was confirmed by spectrophotometry and agarose gel electrophoresis.
Label
CY3
Label protocol
For microarray hybridizations, labeled cDNAs were prepared from 30 µg 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 this experiment, each of the four biological replicates included two technical replicates.
Hybridization protocol
For microarray hybridizations, labeled cDNAs were prepared from 30 µg 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.
Scan protocol
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).
Data processing
A quality control value of "1" 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 deleting 1% of the most intense negative/empty probe values). Intensity values of control spots (a total of 1455 spots including 3X SSC, blanks, and mouse) from MA chips were removed before bioinformatics. In this submission, the original "GPR" files include information for the control spots, while the Sample data tables include information only for the spots that were used for data analyses (a total of: 23937). Raw signal intensities of all DNA-containing spots were up-loaded into the GeneMaths XT 5.1 microarray data analysis program (Applied Maths Inc., Austin, TX, USA) for normalization following the "Layer/Normalization/Arrays" path with default settings (Offset: Average; Scaling: Standard deviation), and for statistical analysis and clustering of the dataset. Hybridization intensities were log2 transformed, and arrays were centered and normalized against each other; technical replicates within each biological replicate were averaged.
Manipulation of Temperature and Photoperiod Identifies Molecular Networks Associated with the Para- to Endo-dormant Transition in Leafy Spurge Crown Buds
Data table header descriptions
ID_REF
VALUE
Log2 normalized ratio of channel 1 over channel 2
CH1_SIG_MED
Raw signal intensity of Chip 20 RD+Vern_Rep3_blue
CH1_BKG_MED
Raw background signal intensity of Chip 20 RD+Vern_Rep3_blue
CH2_SIG_MED
Raw signal intensity of Chip 20 Para_Wk12_Rep3_red
CH2_BKG_MED
Raw background signal intensity of Chip 20 Para_Wk12_Rep3_red
