1. Factors Affecting Flowering in the Biennial Crucifer Barbarea verna Brian W. Tague, Kendrah O. Kidd, Brian J. Ferguson, Rebecca W. Todd, Maryn E. Whittles and Erin Davis Department of Biology, Wake Forest University Winston-Salem NC 27109 “Flowering” in these studies is defined as the conversion of a vegetative meristem to a floral meristem, observed at the macroscopic level. Vegetative apexFloral apex Long days promote inflorescence elongation Data derived as in previous figure. In the case of plants that were vernalized for five weeks and grown with eight hours of light, 65% of the plants showed conversion to a floral meristem; only 25% subsequently bolted. Percent of plants flowering (conversion of meristem) Percent of plants with elongated inflorescence 8 h 24 h 5 weeks10 weeks Greenhouse (>12 h) 5 w10 w Day length: Cold treatment:...but GA can replace long photoperiod induction of inflorescence elongation...... and continued exposure leads to “GA-overdose” UntreatedGA-treated Barbarea verna Length of vernalization (weeks) Percent of plants flowering Barbarea verna must receive 5 weeks of vernalization to flower B. verna seeds were grown as above. 5 week old plants (N >30 for each age) were vernalized from 1 to 10 weeks (t = 4  C). Flowering was scored as described. Unvernalized plants had not flowered after 12 months. Longer vernalization leads to a higher percent of the plants flowering. 8 h 24 h 5 weeks10 weeks Greenhouse (>12 h) 5 w10 w Short days delay flowering in B. verna Five week old plants vernalized for 5 or 10 weeks were subjected to 8 h light/16 hr dark or 24 hr light photoperiods and then scored for flowering. Response of greenhouse grown plants (>12 hours of light) is shown for comparison Average days to flower Day length: Cold treatment: Gibberellic acid cannot replace vernalization in B. verna.... GA-treatedMock treated Ten week old B. verna plants (N >20) were sprayed with a 100 mg/L (0.29 mM) solution of GA 3 until dripping, every day for 9 weeks. No conversion to a floral meristem was observed, although GA-treated plants were greener and had elongated petioles. Abstract An exciting avenue to explore in the post-arabidopsis genome world is the application of tools and information developed in arabidopsis to other plants with unique attributes. To this end, our lab is analyzing the signal transduction pathway regulating flowering in biennials. As a model, we have chosen the obligate biennial crucifer Barbarea verna. Like other obligate biennials, B. verna requires an extended cold treatment to flower. We have shown that B. verna is unresponsive to vernalization treatment until it has grown vegetatively for five or more weeks. The vernalization treatment itself must be at least five weeks long to be effective. In addition to the cold treatment, we have determined that B. verna requires long-days for flowering, producing an abortive inflorescence under short days. The long-day requirement for flowering can be replaced by gibberellic acid treatment. Drawing an analogy to work done in arabidopsis and other species, we are asking whether the cold treatment leads to changes in DNA methylation. Treatments with the demethylating agent 5-azacytidine did not lead to early flowering. Additional demethylating agents are currently being tested, and the degree of DNA methylation in cold- and chemical-treated plants is being measured directly. Additionally, we are generating an Agrobacterium-based transformation protocol for B. verna, using both floral dip and root regeneration protocols. Our goal is transform B. verna with constructs known to induce early flowering in Arabidopsis thaliana to analyze the effects of overexpression of these genes in a biennial plant. We hope to use these experiments to “map” the cold requirement in the signal transduction pathway for flowering in the biennial B. verna. Age of plants before vernalization (weeks) Percent of plants flowering Barbarea verna must be five weeks old to respond to vernalization B. verna seeds were germinated in 4-6” pots and grown in a greenhouse (t = 20-26  C). Plants from 1 to 9 weeks old (N >30 for each age) were vernalized for 5 weeks (t = 4  C). Flowering was scored by the macroscopic appearance of floral buds; plants were observed for 3 months after vernalization. Barbarea verna as a model biennial B. verna (also known as Upland Cress or creasy greens) is a monocarpic perennial that requires vernalization to flower. It grows as a rosette, with characteristics similar to A. thaliana. Relatively small Rosette diameter: ~20 cm Inflorescence height ~40 cm Generation time (in lab): 25-30 weeks Large seed count ~200/plant Easy to grow in soil or plates Self-fertile Introduction Our lab is applying the tools and information developed for Arabidopsis thaliana to related crucifers with attributes not found in arabidopsis. We are currently analyzing the signal transduction pathway regulating flowering in biennials. The biennial habit is an important characteristic of many crop and horticultural plants and could be a target for genetic modification. As a model, we have chosen the obligate biennial Barbarea verna (Brassicaceae). Questions:  Must B. verna reach a minimum age before flowering?  How long is the vernalization period needed for flowering?  What is the effect of photoperiod on flowering?  Does the level of genomic DNA methylation influence flowering?  Can B. verna be transformed? Measuring cytosine methylation by thin layer chromatography MspI (CCGG, C m CGG) HpaII (CCGG) dCMP d m CMP Isolate genomic DNA Restrict with MspI or HpaII End-label with  - 32 P-ATP Digest to nucleotides Separate C and m C by TLC Autoradiography Methylation can be measured using methylation-insensitive restriction enzymes Length of vernalization (weeks) Proportion of cytosines methylated Proportion of cytosine methylation during vernalization B. verna plants were vernalized and transferred to the greenhouse. DNA was isolated from newly emerged leaves and analyzed for cytosine methylation by TLC. Individual spots from the TLC plate were quantified in a scintillation counter. Data shown is average of three experiments; bars show standard error. Gray trend line indicates little change in methylation. Conclusions  B. verna: is a suitable model system for studying biennialism must be at least 5 weeks old to respond to vernalization requires  5 weeks of vernalization to flower  Long days can promote inflorescence elongation  GA does not induce flowering in B. verna  Preliminary experiments indicate no role for methylation in the vernalization of B. verna  Meristem conversion and inflorescence development can be uncoupled Ongoing  Transformation: Floral dipping has not been successful Have regenerated plants from root tissue culture Expression of A. thaliana floral pathway genes in B. verna?  Demethylation: Measuring methylation in 5-azaC treated plants HPLC analysis of cytosine methylation 5-azacytidine treatment can cause cytosine demethylation In certain A. thaliana ecotypes and other plant species that respond to vernalization, chemical demethylation using 5-azaC can induce non-vernalized plants to flower earlier than untreated controls. 5-azaC treatment of B. verna does not induce flowering Conditions tested: Application of 5-azacytidine, 10-100  M Application to >5 week old plants Application to germinating seedlings Treatments led to dwarfing and/or necrosis but no flowering Control 50  M 5-azaC Control 10  m 5-azaC 100  m 5-azaC