Functional analysis of a cold responsive gene in beet Omolade, Seun Ph.D 015733800748 seunomolade@yahoo.com Functional analysis of a cold responsive gene in beet Seun Omolade, Andreas Müller2, Christian Jung1and Siegbert Melzer1 1 Plant Breeding Institute, Christian-Albrechts-University of Kiel, Germany 2 Strube Research GmbH & Co. KG, Söllingen, Germany Research questions Fig. 6 Constitutive BvXX expression affects flowering time Results Results What is the putative function of BvXX in beet? Is BvXX a flowering time gene? How does the expression pattern of BvXX vary with developmental growth stage in both annual and biennial sugar beet? Does BvXX respond to light and/or cold induction? Fig. 2 Phylogenetic relationship of BvXX ** (**: α= 0.05) BvXX Introduction Fig. 1 Floral transition in A. thaliana SOC1 FT Flowering Activation and/or stabilization Inhibition and/or degradation Gibberelin pathway FLC Vernalization pathway Autonomous pathway CO Phototperiod pathway GI CDF1,2,3,5 Circadian clock Ambient Temperature Age pathway Developmental phases LFY AP1 Fig. 3 BvXX expression oscillated in response to light/dark cycles Fig. 7 BvXX disturbs the expression of the core clock gene AtCCA1 Omolade pers. Commun. BvXX/BvGAPDH ZT[h] 1 (LD)/18°C ZT [h] CCA1/IPP2 The initiation of flowering is an important evolutionary and adaptive strategy in plants (Amasino and Micheals, 2010). Genetic analysis in Arabidopsis showed that flower initiation is under the control of both environmental cues (photoperiod through the clock, light) and endogenous pathways. Evidence of conservation and divergence of regulatory functions in these pathways between Arabidopsis and other crops, like sugar beet (Beta vulgaris). The control of flowering in beet is controlled by several already identified genes. BOLTING TIME CONTROL 1 (BTC1), a Pseudo response regulator (PRR) gene, is the photoperiod determinant in beet (Pin et al.,2012) like CONSTANS (CO) in Arabidopsis (Searles and Coupland, 2004; Pose et al., 2012). Another PRR gene was also identified- termed BvXX. Evidence shows that BTC1 is not part of the clock in beet. Fig. 4 BvXX responds to cold Conclusion At 45d in the cold (T4), the transcript level of BvXX increased about 13 fold in annual and biennial beets, and dropped after additional six weeks in the cold (T5). On return to long days at 20°C 24h later (T6), the expression level remained at the initial expression levels while it increased again dramatically after three weeks at 20°C (T7). BvXX is diurnally and developmentally regulated and responds to cold. Transgenic experiment showed BvXX can distort the diurnal rhythm of CCA1 in Arabidopsis and causes late flowering phenotype. References Amasino, R.M., and S.D. Michaels, 2010: The timing of flowering. Plant Physiol 154, 516-20. Pose, D., L. Yant, and M. Schmid, 2012: The end of innocence: flowering networks explode in complexity. Curr Opin Plant Biol 15, 45-50. Pin, P.A., W. Zhang, S.H. Vogt, N. Dally, B. Buttner, G. Schulze-Buxloh, N.S. Jelly, T.Y. Chia, E.S. Mutasa-Gottgens, J.C. Dohm, H. Himmelbauer, B. Weisshaar, J. Kraus, J.J. Gielen, M. Lommel, G. Weyens, B. Wahl, A. Schechert, O. Nilsson, C. Jung, T. Kraft, and A.E. Müller, 2012: The role of a pseudo-response regulator gene in life cycle adaptation and domestication of beet. Curr Biol 22, 1095-101. Searle, I., and G. Coupland, 2004: Induction of flowering by seasonal changes in photoperiod. EMBO J 23, 1217-22. Methods cDNA of BvXX was synthesized, cloned and prepared for further analysis. Phylogenetic analysis was done with the generated BvXX sequence and other downloaded sequences. Expression analysis of BvXX in response to light, at different developmental stages, cold at 6 different time points; one day before cold treatment (T2), 24h of cold treatment (T3), 45d (T4) and 90d (T5) after begin of cold treatment, one (T6) and 90d (T7) after cold treatment. Constitutive expression of BvXX on flowering time (measured as Rosette Leaf Number RLN) and on diurnal regulation of AtCCA1 clock gene. Fig. 5 Developmental expression of BvXX * Flowering stage Seun is currently seeking job placement!