Identification and Molecular Characterization of Circadian Clock Associated Transcription Factors in Rice.

1. Identification and Molecular Characterization of Circadian Clock Associated Transcription Factors in Rice. By Matthew Geniza Mentors: Dr. Todd Mockler and Dr. Sergei Filichkin

2. Importance of Findings • To provide a foundation of the molecular mechanisms that determine plant adaptations depending on their environment. • To increase the basic knowledge of how transcription factors relate to phase-specific gene expression depending on the external conditions. • If Myb-10 is a clock gene, over expressing or suppressing it alters the expression of other clock or clock-regulated genes.

3. The plant circadian clock - at least three interlinked loops PRR5PRR7 PRR9 OUTPUT INPUT flowering CCA1 germination LHY light growth temperature X YGI photosynthesis metabolism TOC1 LUX Plants integrate daily temperature and light information into the circadian clock in order to gauge the time of day and season, and thus coordinate biological processes appropriately for the environment.

4. Background • Plants have evolved an endogenous circadian clock • Internal biological processes are synchronized with changes in the environment. • RNA transcripts of diurnal and circadian regulated genes peak at certain times in the day and night. • Understanding the regulation of gene expression is important in the event of major climate changes

5. Why use rice? • Rice is crop plant that serves as a stable food for a majority of the world. • Rice has a sequenced and annotated genome

6. Questions to ask • Is Myb-10 a clock gene? Does it control oscillations of mRNAs of other rice genes? Does it affect the phases of other clock genes? • Will phase shifting caused by Myb-10 mis-expression changes in phenotype, i.e. growth rate, flowering timing, and ultimately – seed production? • If yes, then Myb-10 is a clock-associated regulator.

7. Cycling of rice Myb10-like TFs in Arabidopsis and poplar 1.2 Putative Myb-like TF AT5G05090 A. thaliana Rice Myb10 GeneID: Os05g34110 Poplar 1 0.8 Normalized Expression 0.6 0.4 0.2 0 0 4 8 12 16 20 24 28 32 36 40 44 Phase http://diurnal.cgrb.oregonstate.edu/

8. Hypothesis • If the Myb-10 gene is a core clock gene, then it will change the oscillations of other core clock genes. • If the Myb-10 gene is NOT a core clock gene, then it will change oscillations of other diurnally regulated genes, but not core clock genes.

9. Experimental Objectives • To identify diurnally regulated genes in rice that are affected by over expression or silencing of a specific circadian clock regulated transcription factor. • To identify cis elements in the promoter region of Myb-10 which regulate its diurnal oscillations.

10. Methods • Rice plants are grown in 12 hours of light and 12 hours of dark cycle for 7 days. This is followed by 60 hours of continuous light. • Tissue samples from plants are collected every 4 hours during the continuous light period. • RNA isolation • Bioanalyzer • Assayed by RT PCR to determine cycling profiles of diurnally regulated gene targets

11. Methods continued • Microarray • The cycling of transcripts of several clock genes (such as CIRCADIAN CLOCK ASSOCIATED1) is studied in the wild type rice and transgenic plants expressing or suppressing Myb-10.

12. Bioanalyzer RIN 10.0 RIN 7.0

13. Gel Image Bioanalyzer

14. RT-PCR Indica LDHC CCA1 Diurnal LDHC CCA1 0 4 8 12 16 20 24 28 32 36 40 44 48

15. RT-PCR Indica LDHC PRR95 Diurnal LDHC PRR95 0 4 8 12 16 20 24 28 32 36 40 44 48

16. Microarray • Quantitative data • Measures gene expression • Assays all genes

17. Future Work • Analyze Microarray Data for Indica • Prepare transgenic Myb-10 plants for Microarray • Analyze Microarray Data for Myb-10 plants • Compare Indica and transgenic Myb-10 plants

18. Special Thanks • Dr. Todd Mockler • Dr. Sergei Filichkin • The Mockler Lab • Dr. Kevin Ahern • HHMI