1. Phenotypic Evolution of Circadian Rhythms in Arabidopsis thaliana
Stephanie Wakefield
Advisors:
Cynthia Weinig and Matthew Rubin

2. Circadian Rhythms The Earth rotates on its axis once every 24 hours
Providing predictable cycles of light and temperature
Most organisms exhibit circadian rhythms
Circadian rhythms are endogenous repeating rhythms
Circadian rhythms are “set” by external cues
Many physiological and behavioral characters are controlled by circadian rhythms
Best example in plants is photosynthesis 2

3. Variation in Circadian Rhythms
Clock Output
Time
Time 3

4. Molecular Basis of Circadian Rhythms
Consists of several negative and positive feedback loops
3 main components:
Input
Oscillator
Output
Clock characterized by knock-out mutants (i.e. functional vs non-functional alleles at clock genes)
Little known about natural allelic variation

5. Research Questions
Does the expression of circadian phenotypes depend on germination conditions (i.e. crowding)?
How have circadian phenotypes evolved after five years of selection in the field?
Do circadian phenotypes depend on substrate type?

6. Arabidopsis thaliana: The Real-Live Plant
Member of the mustard family
Related to cabbage, cauliflower, kohlrabi, broccoli, and kale
Useful for environmental studies:
Primarily self-fertilizing
Naturally highly inbred families
Grow replicates of each genotype in each environment.
Short-lived annual
Fitness estimated from fruit number

7. Distribution of Arabidopsis thaliana
Northern norway and sweeden
Southern to Cape Verde Island, newer populations in Kenya/South Africa and has been recently naturalized in the US 7

8. Arabidopsis thaliana Life Cycles
Sep
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Fall Annual
Spring Annual
In Arabidopsis thaliana, most populations are winter annuals where seeds are dispersed in the spring, lie dormant until the fall, when they germinate, then overwinter as rosette and flower the following spring.
This strategy should be favored in most cases because all of vegetative growth is accomplished prior to the reproductive season allowing plants to maximize the acquisition resources while still flowering at the first opportunity and taking advantage of the entire reproductive season.
But in some populations if the risk of overwinter mortality is high, a cohort of spring annuals or rapid cyclers may also be produced. These plants delay germination until the following spring when all of growth and reproduction must be completed before the onset of summer heat. They are smaller and produce fewer fruits but they are the hedge, the fail-safe, the compensation for winter losses.
In yet a third life history option, some fall germinants may flower in autumn and disperse seeds in the winter.

9. Recombinant Inbred Lines
Col:
USA
Unknown:
Europe X F1
(8 more generations)
Each RIL is a novel combination of parental alleles

10. Growth Chamber Project
Recombinant Inbred Lines (RILs) of Arabidopsis thaliana
42 lab lines and 52 field-evolved lines
16 replicates of 94 RILs per treatment (n=3008)
Cold-treated seeds for 4 days
Germinated in growth chamber
Entrained under artificial spring crowded (PAR= µmol/s*m2, R:FR = 0.52) & un-crowded (R:FR = 1.02)

11. Measuring Circadian Phenotypes
RILs contain a reporter construct
Gene LUCIFERASE (bioluminescence production in fireflies) fused to CCR2 (output to the clock)
Image plant for 30 minutes every hour for 100 hours
Determine circadian phenotypes

12. Period differs in Lab lines

13. Phase differs in both sets of lines

14. Amplitude differs in both sets of lines

15. Amplitude differs between sets

16. Circadian Phase does not differ across sets

17. Longer Period= Later Phase

18. Research Questions
Does the expression of circadian phenotypes depend on germination conditions (i.e. crowding)?
How have circadian phenotypes evolved after five years of selection in the field?
Do circadian phenotypes depend on substrate type?

19. Period and Amplitude depend on substrate type

20. Phase does not depend on substrate type

21. Conclusions
Circadian Phase and Amplitude show significant genetic variation within both lab and field-evolved lines
Circadian Period only differs in Lab lines
High levels of phenotypic variation present in lab lines due to new allelic combinations present as a result of RIL creation
Reduced variation in field line possibly due to elimination of unfit genotypes
Amplitude differs across line types

22. Future Research Increase replication
Phenotype circadian traits under un-crowded conditions
Compare UN to CR

23. Acknowledgements
Funding NSF EPSCoR Research Fellowships for Undergraduates Thanks to: Advisor: Cynthia Weinig and Matthew Rubin Planting/Data Collection : Marc Brock, Dean Lorimer, Melissa Kerr