1. By Matthew Geniza Mentors: Dr. Todd Mockler and Dr. Sergei Filichkin

2. 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 INPUT temperature light OUTPUT flowering germination photosynthesis metabolism CCA1 LHY 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. PRR5 PRR7 PRR9 Y GI X growth

4. 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. Rice is crop plant that serves as a stable food for a majority of the world. Rice has a sequenced and annotated genome

6. 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. Putative Myb-like TF AT5G05090 0 0.2 0.4 0.6 0.8 1 1.2 048121620242832364044 A. thaliana Rice Myb10 GeneID: Os05g34110 Poplar Phase http://diurnal.cgrb.oregonstate.edu/ Normalized Expression

8. 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. 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. 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. Microarray The cycling of transcripts of several clock genes (such as CIRCADIAN CLOCK ASSOCIATED 1) is studied in the wild type rice and transgenic plants expressing or suppressing Myb-10.

12. RIN 10.0RIN 7.0

13. Gel Image

14. Indica LDHC CCA1Diurnal LDHC CCA1 0 4 8 12 16 20 24 28 32 36 40 44 48

15. Indica LDHC PRR95Diurnal LDHC PRR95 0 4 8 12 16 20 24 28 32 36 40 44 48

16. Quantitative data Measures gene expression Assays all genes

17. 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. Dr. Todd Mockler Dr. Sergei Filichkin The Mockler Lab Dr. Kevin Ahern HHMI