1. Rhythms of Life: The Plant Circadian Clock
Somers, D.E. (1999). The physiology and molecular bases of the plant circadian clock. Plant Physiol. 121: 9-20.

2. Living on a rotating planet is biologically stressful
Over a 24 hour period there is large variation in environmental conditions including temperature, light intensity, humidity and predator behaviour
Extreme day-night temperature difference: 57 oC (-48 oC to 9 oC, Montana, 1972)
Typical day-night fluctuation: ~10 oC each day (central Japan)
See Kudoh, H. (2016). Molecular phenology in plants: in natura systems biology for the comprehensive understanding of seasonal responses under natural environments. New Phytol. 210: Image: NASA.

3. Interconnected parts of the circadian system
Circadian gating of entrainment and outputs
Environmental Inputs
Gene
Rhythms in:
- transcription
- physiology
- biochemistry
Entrainment
pathways
Output pathways
Circadian
oscillator

4. The circadian oscillator
Most circadian clocks are
transcription-translation feedback loops
Gene A
Gene B
Protein A
Protein B 12 24 36 48
Transcript abundance
The feedback loop results in rhythms of transcript abundance of the two genes
Time (hours)
Gene A
Gene B
Reciprocal feedback loop
Negative feedback step
Speed of biochemical reactions adds a rate constant
Simple biological
oscillator

5. Morning Loop
Evening Complex
The circadian oscillator is a complex network of interlocking feedback loops
Different clock components are expressed at different times of day
The oscillator includes transcriptional and post-transcriptional processes (not all are shown here)
Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: with permission from Elsevier.. Data from DIURNAL database:

6. Several environmental signals entrain the circadian oscillator
Red light
(phytochrome photoreceptors)
Blue light
(cryptochrome photoreceptors)
Sugars produced by photosynthesis
Circadian
oscillator
Temperature fluctuations

7. Circadian clocks regulate plant cells by controlling gene expression
Some circadian clock proteins are transcription factors that regulate sets of genes with a circadian rhythm
Example: a daytime transcription factor
Day
Gene 1 TF
Specific gene promoter sequences may underlie specific circadian phases of transcription TF TF
Gene 2 TF
Gene 3
Night
Gene 1 TF
Gene 2
Gene 3
Covington, M.F., Maloof, J.N., Straume, M., Kay, S.A. and Harmer, S.L. (2008). Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biology. 9: 1-18.

8. CCA1-ox = arrhythmic transgenic line
Plants with a functioning circadian clock that matches the environment grow larger
T20 = 10 h light, 10 h dark
T24 = 12 h light, 12 h dark
T28 = 14 h light, 14 h dark
(wildtype)
Col-0 = wildtype
CCA1-ox = arrhythmic transgenic line
T24 = 12 h light, 12 h dark
From Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: Reprinted with permission from AAAS.

9. The circadian clock controls multiple aspects of plant biology
Molecular Biology:
~30% of the Arabidopsis thaliana transcriptome oscillates with a 24 period
Circadian rhythms of gas exchange
= wildtype
= arrhythmic mutant (CCA1-ox)
Physiology:
Stomatal opening and closing are under the control of the circadian oscillator
Time (h)
Relative transcript abundance
From Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.-S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (2000). Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science. 290: and from Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: Reprinted with permission from AAAS.

10. The circadian clock controls multiple aspects of plant biology
Growth:
Hypocotyl elongation is clock controlled
Development:
Photoperiod is one of the environmental factors controlling flowering
Wildtype
Circadian clock mutant (gigantea)
Plants grown under long days:
Reprinted with permission from from Dowson-Day, M.J. and Millar, A.J. (1999). Circadian dysfunction causes aberrant hypocotyl elongation patterns in Arabidopsis. Plant J. 17: and Amasino, R. (2010). Seasonal and developmental timing of flowering. Plant J. 61:

11. The circadian clock gives plants a fitness advantage
Arabidopsis thaliana mutant lines with endogenous circadian period:
Competition experiments: When the endogenous period matches the external light-dark cycles, plants perform better in terms of:
survival
biomass (dry and fresh weight)
chlorophyll content
Endogenous period
~20 h
~28 h
Environment
(= 20 h)
(= 28 h)
From Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005). Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 309: Reprinted with permission from AAAS.

12. Time-course analysis is used to study circadian rhythms in plants
The plant is then transferred to conditions of constant light (or dark) and temperature, where circadian-regulated biological process will ‘free run’
The plant is first grown in cycles of light and dark
Biological
process
Time (h)
Light
Dark 12 24 36 48
Phase = time of peak relative to
subjective dawn
Period 12 24 36 48 60 72 84 96
Amplitude
Time (h)
The time that would have been dark is referred to as ‘subjective night’, and is sometimes indicated by grey bars on circadian time-courses

13. Non-invasive measurement techniques benefit the study of circadian rhythms
Measurements of a biological property need to be made frequently (e.g. hourly) over several days
Destructive sampling to obtain RNA or protein is inconvenient: substantial quantities of plant material required, working long hours, opportunities for human error
Non invasive and automated measurement techniques have been developed
Destructive sampling is sometimes essential to monitor rhythms of transcripts, proteins or metabolites.

14. Studying circadian rhythms: Bioluminescence imaging
Expression in plants of an enzyme from fireflies called luciferase causes plants to emit light when provided with the substrate luciferin
Luciferase bioluminescence imaged from Arabidopsis seedlings
Placing LUCIFERASE under the control of a promoter with a circadian rhythm allows the rhythm to be monitored.
The plant emits circadian rhythms of light that can be detected with a very sensitive camera.
Millar, A.J., Short, S.R., Chua, N.H. and Kay, S.A. (1992). A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell. 4:

15. Carbohydrate degradation at night is temporally controlled
The rate of starch degradation is related to the length of the night, so that the plant only exhausts starch reserves just before the end of the night
cca1/lhy mutants exhaust starch reserves at night and enter starvation
Graf, A., Schlereth, A., Stitt, M. and Smith, A.M. (2010). Circadian control of carbohydrate availability for growth in Arabidopsis plants at night. Proc. Natl. Acad. Sci. USA 107:

16. The oscillator, environmental signalling and metabolism form an integrated network
Central Oscillator
CCA1
PRR7/5/9 GI
TOC1
NAD+
Light
Temperature
Chloroplasts
Photosynthesis
Sugar
Redox
ATP/NAD+
Mitochondria
Redox
ATP/NAD+
Image based on Farré, E.M. and Weise, S.E. (2012). The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15: and Haydon, M.J., Hearn, T.J., Bell, L.J., Hannah, M.A. and Webb, A.A.R. (2013). Metabolic regulation of circadian clocks. Semin. Cell Devel. Biol. 24:

17. Plants are more resistant to herbivory when their circadian rhythms are phased with rhythms of insects
When In Phase the plants
resist herbivore attack
Insects
Plants
Entrainment conditions
Free run (constant dark)
When Out of Phase the plants
are vulnerable to herbivores
Goodspeed, D., Chehab, E.W., Min-Venditti, A., Braam, J. and Covington, M.F. (2012). Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. USA 109:

18. Circadian gating changes the sensitivity of signalling responses at different times of day
Example: Circadian gating of light input into the circadian clock
Conceptual Model:
Experimental Data:
Response
Blue light
Red light
Strong response
to light
Identical
light
stimulus
Gate
open
Very weak response to light
Gate
closed
Figure reprinted from Hotta, C.T., Gardner, M.J., Hubbard, K.E., Baek, S.J., Dalchau, N., Suhita, D., Dodd, A.N. and Webb, A.A.R. (2007). Modulation of environmental responses of plants by circadian clocks. Plant Cell Environ. 30: , redrawn from Covington, M.F., Panda, S., Liu, X.L., Strayer, C.A., Wagner, D.R. and Kay, S.A. (2001). ELF3 modulates resetting of the circadian clock in Arabidopsis. Plant Cell. 13:

19. The external coincidence model explains photoperiodic induction of flowering time in long days
= CONSTANS
= FT
First proposed by Bünning (1936). Model redrawn from Imaizumi, T. and Kay, S.A. Photoperiodic control of flowering: not only by coincidence. Trends Plant Sci. 11:

20. CO induces FT expression, which stimulates the floral transition
A molecular model to explain photoperiodic control of flowering time in Arabidopsis
In long days the peak of CO mRNA is during the light, so the CO protein can accumulate
The expression of CO is controlled by the circadian clock, with peak expression ~12 hours after dawn
CO protein is unstable in the dark due to COP1 activity so it doesn’t accumulate and FT is not induced
CO induces FT expression, which stimulates the floral transition
Model redrawn from Imaizumi, T. and Kay, S.A. Photoperiodic control of flowering: not only by coincidence. Trends Plant Sci. 11:

21. The circadian clock and photoperiodic flowering pathways are broadly conserved
Cockram, J., Jones, H., Leigh, F.J., O'Sullivan, D., Powell, W., Laurie, D.A. and Greenland, A.J. (2007). Control of flowering time in temperate cereals: genes, domestication, and sustainable productivity. J Exp. Bot. 58: by permission of Oxford University Press.

22. Circadian clock genes are associated with agronomic traits
Species
QTL/locus
Gene in species
Arabidopsis homologue
Role/Trait
Eudicots
P. sativum
HR/QTL3 HR
ELF3
Circadian clock function, flowering time, light response
Monocots
O. sativa -
OsPRR1
TOC1
OsPRR37
PRR3/PRR7
Flowering time
Ef7/hd17
OsELF3-1
Light-dependent circadian clock regulation
H. vulgare
Ppd-H1
HvPRR37
T. aestivum
Ppd-D1
PRR3/7
Z. mays
ZmGI1 GI
Flowering time and growth regulation
Table based on Bendix, C., Marshall, Carine M. and Harmon, Frank G. (2015). Circadian clock genes universally control key agricultural traits. Mol. Plant. 8:

23. Summary of current understanding of circadian rhythms in plants
Circadian rhythms are molecular time keeping mechanisms that synchronize multiple processes with 24 hour light-dark cycles
The circadian oscillator is a complex feedback loop primarily based on rhythms of gene expression
Investigating circadian rhythms requires novel experimental approaches to capture temporal dynamics
The circadian clock controls metabolism and key developmental transitions
The circadian clock is broadly similar in crop plants, and represents a target for agronomic optimization

24. There are many big questions left in plant circadian biology
Is the oscillator specialized in different cell types, and do these oscillators communicate with each other?
How does plant circadian regulation contribute to ecosystem dynamics?
Can we use our knowledge of circadian biology to increase crop production?
What are the molecular bases of circadian gating?
How did circadian oscillators evolve?