rhythms of life: the plant circadian clock...cell entrainment pathways synchronize the oscillator...

© 2016 American Society of Plant Biologists

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.

http://www.plantphysiol.org/content/121/1/9.full




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 behavior

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: 399-412. Image: NASA.

• Extreme day-night temperature

difference: 57 oC (-48 oC to 9 oC,

Montana, 1972)

• Typical day-night fluctuation:

~10 oC each day (central Japan)

http://onlinelibrary.wiley.com/doi/10.1111/nph.13733/abstract



https://commons.wikimedia.org/wiki/File:Earth_Eastern_Hemisphere.jpg


Circadian clocks are biological

oscillators with a ~24 hour period

Lig

ht-

Dark

cycle

s

Continuous

da

rkne

ss

- Higher levels of wheel running activity at night

- In continuous darkness these rhythms persist but with a

~ 23 hour period Figure from Li, J.-D., Burton, K.J., Zhang, C., Hu, S.-B. and Zhou, Q.-Y. (2009). Vasopressin receptor V1a regulates circadian rhythms of locomotor activity

and expression of clock-controlled genes in the suprachiasmatic nuclei. Am. J. Physiol. 296: R824-R830, used with permission. Image source: Mylius.

Inactive Active

http://ajpregu.physiology.org/content/296/3/R824



https://commons.wikimedia.org/wiki/File:Phodopus_sungorus_-_Hamsterkraftwerk.jpg


Circadian clocks control many

aspects of human physiology

Image source: Addicted04

22.30 Bowel movements suppressed Deepest sleep

21.00 Melatonin secretion starts

19.00 Highest body

temperature

17.00 Greatest muscle

strength

15.30 Fastest reaction time

14.30 Best coordination

06.45

Rise in blood pressure

04.30

Lowest body temperature

02.00

Deepest sleep

Melatonin secretion stops

07.30

High alertness

10.00

18.30 Highest blood

pressure

https://en.wikipedia.org/wiki/Circadian_rhythm_sleep_disorder/media/File:Biological_clock_human.svg


Plant circadian biology has a long

history

Illustration of ‘sleep’ movements in

Medicago, from Charles Darwin (1880) ‘The

Power of Movement in Plants’

Image sources: H. Zell, Charles Darwin “Power of Movement in Plants”

De Marian (1729) ‘Observation

botanique’ of Mimosa pudica:

“sensitive to the Sun and daylight:

the leaves & their peduncles fold

themselves away & contract around

sunset, in the same way they do

when the Plant is touched or

shaken.”

https://commons.wikimedia.org/wiki/File:Mimosa_pudica_002.JPG

https://en.wikipedia.org/wiki/Nyctinasty/media/File:Fig139Movement_of_Plants.png


Architecture of the circadian clock


Principles of operation of plant

circadian clocks

Aspect of the Circadian System

Biological Function

Circadian oscillator Generate a rhythm with a ~24h period within the cell

Entrainment pathways Synchronize the oscillator with the external time of day so that the clock stays accurate

Output pathways Communicate temporal information from the oscillator to other parts of the cell

Circadian gating Adjust the sensitivity of entrainment and output pathways depending on the time of day


Interconnected parts of the

circadian system

Circadian

oscillator

Entrainment

pathways

Output

pathways

Gene

Rhythms in:

- transcription

- physiology

- biochemistry

Environmental

Inputs

Circadian gating of

entrainment and outputs


The circadian oscillator

Most circadian clocks are

transcription-translation feedback loops

Gene A Gene B

Protein A The protein encoded by

Gene A activates Gene B

Protein B

The protein encoded by

Gene B represses Gene A


The circadian oscillator

• Reciprocal feedback loop

• Negative feedback step

• Speed of biochemical reactions adds a rate constant

Simple biological

oscillator

0 12 24 36 48

Gene A Gene B

Protein A

Protein B G

ene tra

nscript

abundance The feedback loop results in

rhythms of transcript abundance

of the two genes

Time (hours)

Gene A

Gene B


An early model for the functioning of

the circadian clock in Arabidopsis

From Alabadı́, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001). Reciprocal regulation between

TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293: 880-883. Reprinted with permission from AAAS

• This is one of the first structures

proposed for the plant circadian

clock

• It is an oscillator with activation

and suppression feedback

(compare with previous slide)

• The main genes involved are

TOC1, LHY and CCA1

• The model is out of date (TOC1

actually suppresses CCA1), but

provides an example of

oscillator structure

Gene A

Protein A Gene B

Protein B

Activation

(out of date!)

Suppression

http://science.sciencemag.org/content/293/5531/880




Reciprocal repression between CCA1

and TOC1 at the core of the circadian

clock

From Alabadı́, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the

Arabidopsis circadian clock. Science. 293: 880-883. Reprinted with permission from AAAS. Gendron, J.M., Pruneda-Paz, J.L., Doherty, C.J., Gross, A.M., Kang,

S.E. and Kay, S.A. (2012). Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. Proc. Natl. Acad. Sci. USA 109: 3167-3172.

Overexpression of CCA1 suppresses

circadian oscillations of TOC1

CCA1 and LHY bind to the promoter

of TOC1

Overexpression of TOC1 suppresses

circadian oscillations of CCA1

The CCT domain of TOC1 is required

for it to bind the CCA1 promoter




http://www.pnas.org/content/109/8/3167.abstract




The current model of the circadian

oscillator is a complex network

Evening Complex

Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier..

Morning Loop

Note TOC1 is a

suppressor of

CCA1/LHY

(compare with

previous model)

http://www.cell.com/trends/plant-science/abstract/S1360-1385%2813%2900260-4




Different clock

components

are expressed

at different

times of day

-1

0

1

2

3

4

5

0 4 8 12 16 20 24

No

rmal

ised

tra

nsc

rip

t ab

un

dan

ce

Time (h)

CCA1

PRR9

LUX

• Network models indicate

connections between

components, but lack temporal

information about clock

function

• This example shows how three

clock genes are activated at

different times of day/night

Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier.. Data from DIURNAL database: http://diurnal.mocklerlab.org/

http://www.cell.com/trends/plant-science/abstract/S1360-1385(13)00260-4



http://diurnal.mocklerlab.org/


The circadian clock also includes

post-transcriptional processes

Chromatin remodeling:

-e.g. promoter of TOC1 has a clock-controlled pattern of

histone 3 (H3) acetylation that affects TOC1 expression

Control of protein stability by proteasome:

-e.g. ZTL is involved in dark-dependent degradation of the

TOC1 protein.

Phosphorylation:

-e.g. Casein Kinase 2 (CK2) phosphorylates CCA1 and LHY

Cytosolic signaling molecules:

-e.g. circadian rhythms of cADPR and Ca2+ in the cytosol

regulate the dynamics of the oscillator

See Más, P. (2008) Circadian clock function in Arabidopsis thaliana: time beyond transcription. Trends Cell Biol. 18: 273-281 for review

http://www.cell.com/trends/cell-biology/abstract/S0962-8924%2808%2900119-0




The circadian oscillator is

temperature-compensated

Temperature (oC)

Col-0 wild type

maintains a ~24 period

across a range of

temperatures, i.e. is

temperature

compensated

PRR7/PRR9

knockdown changes

period in response to

temperature change

Salomé, P.A., Weigel, D. and McClung, C.R. (2010). The role of the Arabidopsis Morning loop components CCA1, LHY, PRR7, and PRR9 in temperature compensation. Plant Cell. 22: 3650-3661;

Nagel, D.H., Pruneda-Paz, J.L. and Kay, S.A. (2014). FBH1 affects warm temperature responses in the Arabidopsis circadian clock. Proc. Natl. Acad. Sci. USA 111: 14595-14600.

PRR7, PRR9 and FBH1 have roles in temperature compensation

28 oC

Period (

h)

Period (

h)

Wild type FBH1-ox FBH1-ox

22 oC

http://www.plantcell.org/content/22/11/3650.abstract








Why is entrainment required?

The time of dawn and dusk is different every single day

Time of sunrise

Time of sunset

Day of year

Tim

e o

f d

ay (

24

h c

loc

k)

Location: Bristol, UK

51.4500° N, 2.5833° W

02:00

04:00

06:00

08:00

0 100 200 300 400

14:00

16:00

18:00

20:00

0 100 200 300 400


Why is entrainment required?

The period of the circadian oscillator is approximately

24 h and there is natural variation between plants

Circadian period (h)

Me

asu

rem

en

ts

Reprinted from Swarup, K., Alonso-Blanco, C., Lynn, J.R., Michaels, S.D., Amasino, R.M., Koornneef, M. and Millar, A.J. (1999). Natural allelic variation identifies new genes in the Arabidopsis circadian system. Plant J. 20: 67-77.

• Measurements of

circadian period in

three wild type strains

of Arabidopsis thaliana

• There is variation

within and between

strains, but all have a

period of approximately

24 hours

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.1999.00577.x/abstract




Several environmental signals

entrain the circadian oscillator

Circadian

oscillator

Red light

(phytochrome photoreceptors)

Blue light

(cryptochrome photoreceptors)

Sugars produced by photosynthesis

Temperature fluctuations


Phytochromes and cryptochromes

provide light input to the circadian

clock

cry1 mutant

cry2 mutant

Fluence rate of blue light

(µmol m-2 s-1)

Wild type

Wild type

From Somers, D.E., Devlin, P.F. and Kay, S.A. (1998). Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science. 282: 1488-1490 Reprinted with permission from AAAS

Fluence rate of red light

(µmol m-2 s-1)

phyA mutant

Wild type

phyB mutant

Wild type

http://science.sciencemag.org/content/282/5393/1488.abstract




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

Gene 2

Gene 3

TF

Gene 1 TF

TF

TF

Example: a

daytime

transcription

factor

Gene 1

Gene 2

Gene 3

TF

Day

Night


Specific gene promoter sequences

may underlie specific circadian

phases of transcription

• These cis elements occur

with high frequency in

promoters of transcript sets

with certain circadian

phases

• Indicates that the circadian

clock regulates different

subsets of genes with

different circadian phases

through particular clock-

controlled promoter motifs

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.

https://genomebiology.biomedcentral.com/articles/10.1186/gb-2008-9-8-r130




The importance of circadian rhythms

in plant biology


T20 = 10 h light, 10 h dark



(wildtype)

Plants with a functioning circadian

clock that matches the environment

grow larger

Col-0 = wildtype

CCA1-ox = arrhythmic transgenic line


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: 630-633. Reprinted with permission from AAAS.

http://science.sciencemag.org/content/309/5734/630.full




The circadian clock controls

multiple aspects of plant biology Molecular Biology: ~30% of the Arabidopsis

thaliana transcriptome oscillates

with a 24 period

Time (h) Rela

tive tra

nscript

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: 2110-2113 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: 630-633. Reprinted with permission from AAAS.

Circadian

rhythms of gas

exchange

= wildtype

= arrhythmic

mutant (CCA1-

ox)

Physiology: Stomatal opening and closing are

under the control of the circadian

oscillator








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: 63-71 and Amasino, R. (2010). Seasonal and developmental timing of flowering. Plant J. 61: 1001-1013.

http://onlinelibrary.wiley.com/wol1/doi/10.1046/j.1365-313X.1999.00353.x/full







The circadian clock gives plants a

fitness advantage

~20 h ~28 h

(= 20 h)

(= 28 h)

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

Arabidopsis thaliana mutant lines

with endogenous circadian period:

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: 630-633. Reprinted with permission from AAAS.

Endogenous

period

Environment





Investigating the circadian clock in

the laboratory


Time-course analysis is used to

study circadian rhythms in plants

The plant is first grown in

cycles of light and dark

Bio

logic

al

pro

cess

Time (h)

Light Dark Light Dark

0 12 24 36 48

The plant is then transferred to

conditions of constant light (or dark) and

temperature, where circadian-regulated

biological process will ‘free run’

The time that would have been dark is referred to as ‘subjective night’, and is

sometimes indicated by grey bars on circadian time-courses

0 12 24 36 48 60 72 84 96 Time (h)


Circadian rhythms have a number of

measurable properties

0 12 24 36 48 60 72 84 96

Bio

logic

al pro

cess

Period

Amplitude

Phase = time of peak relative to

subjective dawn

Time (h)

Period = time to complete one full cycle

Phase = the time at which a particular point of cycle occurs (e.g. the peak)

Amplitude = the displacement of the oscillation from the center point

‘Subjective dawn’ occurs

every 24h after lights on


Common methods for studying circadian

rhythms: Transcript analysis

Individual transcripts

can be monitored

Circadian rhythms of the whole

transcriptome can be studied with

microarrays or RNA sequencing or

Photosystem I and II

transcripts

Light harvesting complex

transcripts

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: 2110-2113. Reprinted with permission from AAAS.

This transcriptome

analysis found that

many photosynthesis

genes have circadian

rhythms





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, long

working 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


Common methods for studying

circadian rhythms: Leaf movement

From Hicks, K.A., Millar, A.J., Carré, I.A., Somers, D.E., Straume, M., Meeks-Wagner, D.R. and Kay, S.A. (1996). Conditional circadian dysfunction of the

Arabidopsis early-flowering 3 mutant. Science. 274: 790-792. Reprinted with permission from AAAS. Image credits: Vojtěch Zavadil; K.Hubbard unpublished

Leaf positio

n (

pix

els

)

8 d

ays o

ld

(sub

j. d

ay)

8 d

ays o

ld

(sub

j. n

igh

t)

9 d

ays o

ld

(sub

j. d

ay)

0 24 48 72 96 120 144

Automated video imaging and

image analysis allows

quantification of leaf movements

Some plants have a

pulvinus at the base

of the leaf which

drives movement of

the leaves

In Arabidopsis, leaf

movements are

part of rhythmic

patterns in growth




https://commons.wikimedia.org/wiki/File:3977-Gleditsia_triacanthos-pulvinus-Arb.Brno-10.12.jpg



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 Luciferin

O2

ATP

Light

Oxyluciferin

AMP

LUCIFERASE transgene Plant genome



circadian rhythms:


•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.

Luciferase bioluminescence

imaged from Arabidopsis

seedlings

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: 1075-1087.






circadian rhythms:


From Millar, A., Carre, I., Strayer, C., Chua, N. and Kay, S. (1995). Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science. 267: 1161-1163. Reprinted with permission from AAAS.

• LUCIFERASE is placed

under the control of the

rhythmic CAB2 promoter

• Around 8000 seedlings

from a mutagenized

population were tested, to

identify components of the

circadian clock

• This allowed identification

of circadian clock

components (here, TOC1)

Mutant with short

circadian period (toc1-1)

Wild type

seedlings

http://science.sciencemag.org/content/267/5201/1161.long




Advanced methods for studying

circadian rhythms:

Rhythms in individual tissues

1. Use a super sensitive camera with close up lens to monitor luciferase

Bio

lum

inescence f

rom

indiv

idual pix

els

Luciferase bioluminescence

from single Arabidopsis leaf

Wenden, B., Toner, D.L.K., Hodge, S.K., Grima, R. and Millar, A.J. (2012). Spontaneous spatiotemporal waves of gene expression from biological clocks in the leaf. Proc. Natl. Acad. Sci. USA. 109: 6757-6762.






circadian rhythms:

Rhythms in individual tissues 2. Split luciferase into two fragments, and give

one a tissue-specific gene promoter

This half of luciferase is only

expressed when the clock promoter

is active

This half of luciferase is only

expressed in the specific tissue type

The complete protein is only

assembled when both

promoters are active

Reprinted by permission from Macmillan Publishers Ltd: Endo, M., Shimizu, H., Nohales, M.A., Araki, T. and Kay, S.A. (2014). Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515: 419-422.

http://www.nature.com/nature/journal/v515/n7527/full/nature13919.html





circadian rhythms:

Rhythms in individual tissues

One half of luciferase: Vascular SUC2 promoter

One half of luciferase: Circadian TOC1 promoter

This was used to show the circadian clock

in vascular tissue is dominant to that of

other cell types in leaves 1 mm

2. Split luciferase into two fragments, and give

one a tissue-specific gene promoter

Reprinted by permission from Macmillan Publishers Ltd: Endo, M., Shimizu, H., Nohales, M.A., Araki, T. and Kay, S.A. (2014). Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature. 515: 419-422.






circadian rhythms: The value of

mathematical modeling

How do you unravel how this functions? It’s totally non-intuitive!

Large number of interconnected clock components

Multiple feedback loops, both positive and negative

Each protein and transcript has its own unique rate of synthesis and breakdown

Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier..






circadian rhythms: The value of

mathematical modeling

Building mathematical simulations of the clock has:

• Provided new information about how clock

components interact

• Provided explanations for why the clock is so complex

• Provided new information about how the clock is

entrained to the environment

• Demonstrated the power of mathematical and

systems biology approaches for biological research


The circadian clock and plant

metabolism


The circadian clock has extensive

control of plant metabolism

Almost all metabolic

pathways include at

least one enzyme that is

under circadian

transcriptional control

• Each square = One circadian-

regulated transcript

• Color of square = phase of

expression

Reprinted from Farré, E.M. and Weise, S.E. (2012). The interactions between the circadian clock and primary metabolism. Curr. Opin. Plant Biol. 15: 293-300 with permission from Elsevier.

http://www.sciencedirect.com/science/article/pii/S1369526612000258




Many metabolic pathways have

physiologically appropriate phases

of maximal transcript abundance

Data from DIURNAL database: http://diurnal.mocklerlab.org/

0.4

0.6

0.8

1

1.2

1.4

1.6

0 12 24 36 48

Rel

ativ

e Ex

pre

ssio

n L

evel

Time in continuous light (h)

Chlorophyll Biosynthesis

0

0.5

1

1.5

2

0 12 24 36 48

Rel

ativ

e Ex

pre

ssio

n L

evel

Time in continuous light (h)

Starch Catabolism

Dawn Dusk Dawn Dawn Dusk

Subjective time of day:

Chlorophyll biosynthesis

genes peak just before dawn

to anticipate light availability

Starch catabolism genes peak

around dusk

http://diurnal.mocklerlab.org/


Circadian clock mutants have

altered metabolite levels in light-

dark cycles Data shown for a prr9/7/5

triple mutant grown in

12h light: 12h dark cycles

Increase in Citric Acid

Cycle intermediates

Increase in Shikimate

suggests changes in

secondary metabolism

Fukushima, A., Kusano, M., Nakamichi, N., Kobayashi, M., Hayashi, N., Sakakibara, H., Mizuno, T. and Saito, K. (2009). Impact of

clock-associated Arabidopsis pseudo-response regulators in metabolic coordination. Proc. Natl. Acad. Sci. USA 106: 7251-7256.





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

12hL:12hD cycles (normal growth condition)

Early night imposed

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: 9458-9463.





cca1/lhy mutants exhaust starch

reserves at night R

ela

tive E

xpre

ssio

n L

evel

wildtype

cca1-11/lhy-21

cca1/lhy mutants:

-Accumulate 20% less

starch than wildtypes

-Degrade starch 35% faster

than wildtypes at night

-Exhaust starch reserves 3-

4 hours before the end of

the night

-Express starvation genes

before the end of the night

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: 9458-9463.





The chloroplast has circadian

rhythms of gene expression that are

controlled by the nucleus

psbD = Chloroplast gene

SIG5 = Nuclear gene

chloroplast

cytosol chloroplast

Circadian

oscillator

SIG5 TF

Gene SIG5

SIG5 imported

into chloroplast

From Noordally, Z.B., Ishii, K., Atkins, K.A., Wetherill, S.J., Kusakina, J., Walton, E.J., Kato, M., Azuma, M., Tanaka, K., Hanaoka, M. and Dodd, A.N.

(2013). Circadian control of chloroplast transcription by a nuclear-encoded timing signal. Science. 339: 1316-1319. Reprinted with permission from AAAS.





Primary metabolites regulate the

activity of the circadian oscillator

Dalchau, N., Baek, S.J., Briggs, H.M., Robertson, F.C., Dodd, A.N., Gardner, M.J., Stancombe, M.A., Haydon, M.J., Stan, G.-B., Gonçalves, J.M. and Webb, A.A.R. (2011). The

circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose. Proc. Natl. Acad. Sci. USA 108: 5104-5109.

Application of external

sucrose restores circadian

rhythms in wildtype

seedlings grown in the dark

This is dependent on the

presence of the oscillator

component GIGANTEA





The oscillator, environmental

signalling and metabolism form an

integrated network

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: 293-300 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: 414-421.

Central Oscillator

CCA1

PRR7/5/9

GI

TOC1

Metabolism Environmental

Signalling

Chloroplasts

Photosynthesis

Sugar

Redox

ATP/NAD+

Mitochondria

Redox

ATP/NAD+

NAD+

Light

Temperature








The circadian clock regulates

production of volatile compounds

Petunias use the emission

of volatile compounds to

attract nocturnal pollinators

such as hawkmoths

There are circadian rhythms of

volatile emissions:

μg

em

itte

d/g

fre

sh

we

igh

t/h

r

Fenske, M.P., Hewett Hazelton, K.D., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A. and Imaizumi, T. (2015). Circadian clock gene

LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proc. Natl. Acad. Sci. USA 112: 9775-9780.





The circadian clock regulates

production of volatile compounds Many genes required for volatile production are under transcriptional

control of the oscillator:

Volatile compounds

Circadian transcriptional

control







Transgenic overexpression of PvLHY

abolishes nocturnal production of

volatiles

W115 = wildtype line

#37* = PvLHY overexpressing line

Circadian mutants of Petunia do not

emit volatile compounds at night







Plants are more resistant to herbivory

when their circadian rhythms are

phased with rhythms of insects

Insects

Plants

Insects

Plants

When In Phase the plants

resist herbivore attack

When Out of Phase the plants

are vulnerable to herbivores

Entrainment

conditions

Free run

(constant dark)

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: 4674-4677.





Plants produce jasmonates during

the subjective day to deter

herbivores Insects feed during

the subjective day

Jasmonates accumulate

during the subjective day

Jasmonate accumulation induces herbivore defense mechanisms

e.g. production of toxic secondary metabolites

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: 4674-4677.





The circadian clock provides timing

information to control photoperiodic

flowering


4

6

8

10

12

14

16

18

Sep

Oct

No

v

Dec Jan

Feb

Mar

Ap

r

May Jun

Jul

Au

g

Day

len

gth

(h

ou

rs)

Cambridge, UK

Many plants use photoperiod as a

signal to sense seasonal changes

Winter = short

photoperiod

(~7 h of light)

Summer = long

photoperiod

(~15 h of light)

Wheat (Triticum

aestivum) flowers in the

spring when the days

are getting longer

Image source: anthere

https://commons.wikimedia.org/wiki/File:WheatFlower1.jpg


Photoperiod sensitive plants induce

flowering under either long or short

days Long Day Plants

e.g., Wheat

Short Day Plants

e.g., Rice

24 hours

Critical night length

24 hours

Critical night length

Flash Flash

(Note: flowering of some species e.g. tomato is photoperiod insensitive; these are referred to as day-neutral plants)


Flowering time is a highly regulated

event which centers on FLOWERING

LOCUS T (FT)

Induction of FT

expression in leaves FT protein

moves to the

shoot apical

meristem via

the phloem

Floral integrator

genes induced

in meristem, and

flowering is

initiated

Photoperiod

Age

(autonomous

pathway)

Cold winters

(vernalization

pathway)

Gibberellin


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: 550-558.

https://en.wikipedia.org/wiki/Erwin_B%C3%BCnning





0

5

10

15

20

25

30

35

40

45

50

Long Days(16hL: 8hD)

Short Days(8hL: 16hD)

No

. of

leav

es

at f

low

eri

ng

WT (Ler)

co-2

The zinc finger transcription factor

CONSTANS is the circadian

dependent regulator

Zn

ion

Data: K. Hubbard (unpublished). Image source: Thomas Splettstoesser

The zinc finger is a DNA

interaction motif found in all

kingdoms of life CONSTANS is required for

floral induction in long days

in Arabidopsis

https://en.wikipedia.org/wiki/Zinc_finger/media/File:Zinc_finger_DNA_complex.png


A molecular model to explain

photoperiodic control of flowering

time in Arabidopsis

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

In long days the

peak of CO mRNA

is during the light,

so the CO protein

can accumulate

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: 550-558.





The photoperiodic flowering

pathway is broadly conserved

between Arabidopsis and crops

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: 1231-1244 by permission of Oxford University Press.

http://jxb.oxfordjournals.org/content/58/6/1231.abstract




A small change to the model also

explains flowering in short day

plants such as rice

Long Day Plants:

CO activates FT expression Short Day Plants:

‘CO’ represses ‘FT’ expression

Model redrawn from Song, Y.H., Shim, J.S., Kinmonth-Schultz, H.A. and Imaizumi, T. (2015). Photoperiodic flowering: Time measurement mechanisms in leaves. Annu. Rev. Plant Biol. 66: 441-464.

http://www.annualreviews.org/doi/abs/10.1146/annurev-arplant-043014-115555




Circadian gating


Circadian gating: General concept

• The regulation of other cell signaling pathways by

the circadian clock is known as circadian gating

• It is a fundamental way that circadian clocks

regulate plant cells

• During gating, the clock acts as a valve on the

response of the plant to the environment, so the

same environmental cue causes a different strength

response depending on the time of day

• At some times of day the gate is open and the

signal passes through. At other times of day the

gate is closed and the signal cannot pass through


Circadian gating: General concept

General principle: an example of light signaling

Strong response

to light

Response

Very weak

response to

light

Identical

light

stimulus

Gate

open

Gate

closed


Circadian gating acts upon circadian

entrainment and environmental

signaling pathways

1.The circadian clock gates the light and

temperature signals that entrain the

circadian clock

2.The circadian clock gates signaling

pathways that regulate the responses of

plants to the environment


1. Circadian gating of light input to

the circadian clock • If the circadian clock

responded identically to

light at every time of day, it

would be reset to dawn

continuously and be unable

to provide a measure of

time

• The clock regulates its own

sensitivity to light, so the

way it responds to light

depends on the time of day Blue light

Red light

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: 333-349, 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: 1305-1316.

http://onlinelibrary.wiley.com/wol1/doi/10.1111/j.1365-3040.2006.01627.x/abstract







2. Circadian gating of environmental

response pathways Example: The circadian clock gates responses of plants to a cold environment

Fowler, S.G., Cook, D. and Thomashow, M.F. (2005). Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol. 137: 961-968.

*** *** *** *** *** *** ---- ---- ----

*** ----

More sensitive to cold when transferred to cold at this time

Less sensitive to cold when transferred to cold at this time

CB

F2 t

ranscript

level

http://www.plantphysiol.org/content/137/3/961.abstract




3. Circadian gating of environmental

response pathways Example: The circadian clock gates shade avoidance in Arabidopsis

Time in constant light (h)

Change in h

ypoco

tyl le

ngth

(%

)

Hypocotyl

• Seedling hypocotyls elongate rapidly in

the shade to overtop their competitors

• Seedlings are most sensitive to

simulated shade around subjective dusk

Reprinted by permission from Macmillan Publishers Ltd: Salter, M.G., Franklin, K.A. and Whitelam, G.C. (2003). Gating of the rapid shade-avoidance response by the circadian clock in plants. Nature. 426: 680-683.





The potential for crop improvement

using circadian-dependent traits


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 ELF3 Light-dependent circadian clock regulation

H. vulgare Ppd-H1 HvPRR37 PRR3/PRR7 Flowering time

T. aestivum - Ppd-D1 PRR3/7 Flowering time

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: 1135-1152.

http://www.cell.com/molecular-plant/abstract/S1674-2052%2815%2900171-9




Case study 1: A slower clock was

selected for during the

domestication of tomato

Reprinted by permission from Macmillan Publishers Ltd: Muller, N.A., Wijnen, C.L., Srinivasan, A., Ryngajllo, M., Ofner, I., Lin, T., Ranjan, A., West, D., Maloof, J.N., Sinha,

N.R., Huang, S., Zamir, D. and Jimenez-Gomez, J.M. (2016). Domestication selected for deceleration of the circadian clock in cultivated tomato. Nat Genet. 48: 89-93.

• Two QTLs were identified,

one of which mapped to a

homologue of an

Arabidopsis light signaling

protein (EID1)

• Plants with the phase delay

mutation were late

flowering and had higher

chlorophyll content in long

days, indicating a

competitive advantage

http://www.nature.com/ng/journal/v48/n1/full/ng.3447.html




Case study 2: The role of Ppd-H1 in

photoperiodic responses of barley

Image source: Craig Nagy

Barley (Hordeum vulgare):

- Long-day plant, i.e., it requires

day lengths in excess of a critical

minimum to flower.

- There are also varieties that are

insensitive to day length

Photoperiod

sensitive

‘Igri’

Photoperiod

insensitive

‘Triumph’

x

Mapping

population

generated

Ppd-H1 locus identified

https://commons.wikimedia.org/wiki/File:Korea-Barley-01.jpg


Ppd-H1 is a homologue of the

Arabidopsis PRR7 oscillator gene

At PRR7 is a pseudo-response

regulator that has 50% overall

similarity to Hv Ppd-H1

Phenotypes of

homozygous Ppd-H1 (left),

heterozygous (middle), and

homozygous ppd-H1 (right) plants

From Turner, A., Beales, J., Faure, S., Dunford, R.P. and Laurie, D.A. (2005). The Pseudo-Response Regulator Ppd-H1 provides adaptation to photoperiod in barley. Science. 310: 1031-1034. Reprinted

with permission from AAAS. Reprinted from Hsu, P.Y. and Harmer, S.L. (2014). Wheels within wheels: the plant circadian system. Trends Plant Sci. 19: 240-249 with permission from Elsevier..








Winter barley (Ppd-H1) is

photoperiod sensitive and flowers in

early spring

4

6

8

10

12

14

16

18

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug

Day

len

gth

(h

ou

rs)

Cambridge, UK

Lebanon

Autumn Winter Spring Summer

Sowing Harvest Harvest occurs

before heat of

summer

Flowering is induced when days get

longer than ~13 hours (i.e. in the spring),

resulting in early harvest Winter barley (Ppd-H1)

See 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: 1231-1244.





Spring barley (ppd-H1) is

photoperiod insensitive and flowers

late

4

6

8

10

12

14

16

18

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug

Day

len

gth

(h

ou

rs)

Cambridge, UK

Lebanon


Flowering fails to be induced when

days get longer than ~13 hours (i.e.,

in the spring), therefore crop grows

throughout the summer

Sowing Harvest

Spring barley (ppd-H1)

Sowing in

spring avoids

frost damage

See 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: 1231-1244.





Photoperiod insensitive landraces of

barley are more common in northern

Europe


Harvest Sowing

Sowing Harvest

North = spring barley (ppd-H1)

Insensitive to photoperiod so flowers late

and grows throughout the summer.

Avoids the winter frost

South = winter barley (Ppd-H1)

Sensitive to photoperiod so flowers in the

spring when days get longer.

Avoids the heat of summer.

Spread of barley

northwards from

the fertile crescent

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: 1231-1244 by permission of Oxford University Press.





Summary and Future Perspectives


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


There are many big questions left in

plant circadian biology

What are the molecular bases

of circadian gating?

Is the oscillator specialized in different cell types, and do these oscillators communicate

with each other? Can we use our knowledge of

circadian biology to increase crop

production?

How does plant circadian regulation contribute to

ecosystem dynamics?

How did circadian oscillators evolve?

rhythms of life: the plant circadian clock...cell entrainment pathways synchronize the oscillator...

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