circadian rhythm in plants

1. NAME: CAUVERY H BUGATI
PALB 7253, Sr.MS.c (agri)1

2. HISTORY
PARAMETERS OF CIRCADIAN RHYTHM
CHARACTERISTICS
CRITERIA
CASE STUDIES AND CONCLUSIONS
MODEL OF SIMPLE CIRCADIAN CLOCK
FLOW OF SEMINAR
MOLECULAR BASIS OF CIRCADIAN RHYTHM
2

3. • Most organisms have acquired the capacity to
measure time and use its information to temporally
regulate their biology and coordinate with the
environment in anticipation of coming change.
I need to
protect
myself from
the sun
Its nearly
sunset so I
need to get
ready for cold
Sunrise Sunset
(Kamioka et al., 2016)3

4.  Plants, like all eukaryotes and most prokaryotes, have
evolved sophisticated mechanisms for anticipating
predictable environmental changes that arise due to the
rotation of the Earth on its axis.
 These mechanisms are collectively termed ‘CIRCADIAN
RHYTHMS’.
4

5. HISTORY
Depiction of the flower clock
(eine Blumen-Uhr) designed
by Linneaus.
Left half – (6 AM-12 PM) –
petals – opening.
Right half – (12 PM-6 PM) –
petals – closing. (except
evening primrose, which starts
to open its flowers after 5 PM)
Somers, 1999 5

6. 1729 - Jean Jacques d’Ortus de Mairan – French astronomer
The experimental approach to the study of endogenous biological rhythm.
Experimental material – Mimosa pudica.
1st experimental evidence for the persistence of an endogenous
rhythmicity in the absence of environmental cues.
1832 - Augustin de Candolle – Frenchman. He determined that the free
running period of M. pudica was 22 to 23 h, discernably shorter than 24
h.
1894 - Kiesel - Animal circadian rhythms were first described for pigment
rhythms in arthropods.
1922 - Richter - daily activity in rats to circadian rhythms. 6

7. 1935 - Erwin Bünning - identified two variants of common bean
(Phaseolus vulgaris) that differed in their endogenous period length by 3 h.
The property of circadian rhythms is a genetically based polygenic trait.

 1950s - Franz Halberg of the University of Minnesota
coined the term circadian. "father of American
chronobiology”.
 1971 - Ron Konopka and Seymour Benzer - isolated the first clock
mutant in Drosophila mapped the "period" gene, the first discovered
genetic determinant of behavioural rhythmicity.
horizontal day position and vertical night position.
7

8. 1994 - Joseph Takahashi - discovered the first mammalian
circadian clock mutation (clockΔ19) using mice.
2017 - Jeffrey C. Hall, Michael Rosbash and Michael W.
Young - discoveries of molecular mechanisms controlling the
circadian rhythm in Drosophila.
8

9. The periodic or cyclic phenomena in living organisms that help in
the anticipation and adaptation to solar- and lunar-related rhythms
are called biological rhythms
CHRONOBIOLOGY - Greek word
chronos - time
biology - study or science of life
Infradian
rhythm
Circadian
rhythm
Ultradian
rhythm
Types of rhythms
9

10. Types of rhythms
1. Infradian rhythm 2. Circadian rhythm 3. Ultradian rhythm
• cycles longer than
a day
• roughly 24-hour
cycle shown by
physiological
processes in all the
organisms.
• cycles shorter than
24 hours.
• Eg: circannual or
annual cycles that
govern migration
of birds.
• Eg: leaf movements
in plants.
• Eg: the 90-minute
REM cycle.
10

11. Examples of circadian rhythms in plants
11

12. • It is an external time
• based on a normal 24-hour
cycle.
• It is an internal time
• based on free running
period.
(McClung, 2001)12

13. Circadian clock
• In order to adapt to the alternation
between day and night caused by the
rotation of the earth, many organisms
possess a circadian clock (biological
clock)
• Circadian clock, an internal timer or
oscillator that keeps approximately
24-hour time.
(Kamioka et al., 2016) 13

14. Processes controlled by clock
Sleep/wake cycles in animals
Developmental transitions
in filamentous fungi
The incidence of heart
attacks in human
Hibernation in mammals
Long-distance
migration in butterflies
14

15. DAILY RHYTHMS SEASONAL RHYTHMS
photosynthesis
stem growth
scent emission
flowering in plants
the onset of dormancy
(Harmer, 2009)15

16. Franz Halberg (1959) coined the term
circadian - Latin words ‘‘circa’’ (about)
‘‘dies’’ (day)
Circadian rhythms is endogenous, self
sustaining rhythms with periods of ̴ 24 h,
are driven by an internal circadian clock,
persist under constant environmental
condition. (Dunlap et al., 2004)
ULTRADIAN --- less than 24 hours
INFRADIAN --- more than 24 hours
Circadian Rhythm
16

17. PARAMETER OF CIRCADIAN RHYTHMS
• Form of sinusoidal waves
• Mathematical terms
PERIOD
Defined as time to complete one cycle.
 commonly measured from peak to peak, or trough to
trough, or from any specified phase marker
PHASE
 Phase is the time of day for any given event.
AMPLITUDE
One half the peak-to-trough distance
17

18. ZEITGEBER
By circadian convention, the time of onset of a signal that resets the
clock is defined as zeitgeber (“time giver”) time 0, abbreviated ZT0.
(Harmer, 2009)18

19. Many plant processes are rhythmic
19

20. Where these rhythms are seen …???
CIRCADIAN
CLOCK
CIRCADIAN
RHYTHM
CELLULAR
LEVEL
Changes gene
transcription
WHOLE
ORGANISM
LEVEL
Changes in
activity
(Harmer, 2009)20

21. Intracellular processes
• Gene transcription
• Ca+2 levels
• Some enzyme
activities
Higher level of
organization events
• Rhythms in stomatal
opening
• Leaf movement
• Hypocotyl expansion
Fundamental changes
in plant development
• Photoperiodic control
of flowering
• Onset of bud
dormancy
(Somers, 1999))
21

22. When can we define these
processes as outputs of the
circadian clock rather
than mere responses to
environmental cues ?
22

23. i. Circadian rhythms persist with approximately (but never exactly)
24-hour periodicity after an organism is transferred from an
environment that varies according to the time of day (entraining
conditions) to an unchanging environment (free-running
conditions).
ii.The time of onset of these rhythms can be reset by appropriate
environmental cues, such as changes in light or temperature levels.
CRITERIA
23

24. CHARACTERISTICS
1. Endogenous Clock persist with approximately
(but not exactly) 24-hour periodicity
2. Entrainability
3. Circadian rhythm are temperature compensated
4. The persistence of rhythmicity in the absence of
periodic input
(Somers, 1999)24

25. 1. Endogenous Clock
 Endogenous substances and processes → originate
from within an organism, tissue, or cell.
 The rhythm persists in constant conditions, (i.e., constant darkness)
with a period of about 24 hours.
 The period of the rhythm in constant conditions is called the free-
running period, denoted by the Greek letter τ (tau).
 A rhythm cannot be said to be endogenous unless it has been tested and
persists in conditions without external periodic input.
 In diurnal animals → τ is slightly greater than 24 hours.
 In nocturnal animals → τ is shorter than 24 hours. 25

26. Process by which the clock is synchronized to the outside
world/environment is called as Entrainability.
 Diurnal oscillations in temperature (high/low) or light (light/dark)
are the cues that adjust the circadian system with each cycle.
 Alter the position, or phase, of the oscillation.
 For fully entraining the clock
• Drosophila → a short 15-min light pulse
• Plants → 3 to 4 hr of illumination
2. Entrainability
26

27. The circadian rhythms occur with approximately same periodicity
across a wide range of temperatures.
Most biochemical reactions - Q10 value will be nearly 2 - 3
(approximately double the rate of the process)
In contrast, the Q10 values of circadian rhythms lie between 0.8 and
1.4 (Q10 = 1)
This characteristic allows the circadian system to keep accurate time
even when ambient conditions are cold or hot.
3. Temperature Compensation
27

28. 4. The persistence of rhythmicity in the absence of
periodic input
The primary focus is to understand the mechanism of the circadian
clock.
To identify the components and their interactions that allow the
maintenance of a self-sustained oscillation in a non-periodic
environment.
28

29. Why do plants have clocks?
29

30.  Clock allow organisms to anticipate regular changes in the
environment and synchronize different physiological processes
with each other.
 Clocks likely provide an adaptive advantage by allowing proper
timing of physiology with respect to the environment.
30

31. Model of a Simple Circadian System
(Somers et al., 1998)31

32. The three primary components include:
 an input (entrainment) pathway(s)
 the central oscillator
 an output pathway(s)
The phytochromes (PHY) and the cryptochromes (CRY) are two
classes of photoreceptors known to mediate the first step of the light
entrainment pathway
Interactions among the components (A–D) of the central oscillator
create the autoregulatory negative-feedback loop that generates the
approximately 24-h oscillations.
Three different hypothetical couplings of the central oscillator to
possible output pathways are shown to indicate that differently
phased overt rhythms with the same period (E–G) can arise from a
single pacemaker.
(Somers et al., 1998)32

33. INPUT PATHWAYS
( LIGHT ENTRAINMENT)
 Red-light-sensing PHYs (phytochromes)
 Bluelight-sensing CRYs (cryptochromes)
Although the CRYs and PHYs govern light input, they are also
rhythmic outputs of the clock.
• EARLY FLOWERING 3 (ELF3)
• EARLY FLOWERING 4 (ELF4)
SENSITIVITY TO REDLIGHT REDUCED 1 (SRR1),
• positively regulator of both red and white light.
All exhibit circadian oscillations at the RNA level, though only PHYA,
PHYB and PHYC appear to oscillate at the protein level.
Gardner et al.(2006)
Negatively regulates
light input
33

34. CORE OSCILLATOR
► Consist of elements arranged in interlocking transcriptional
feedback loops.
► The first loop to be described consists of
• TOC1 (TIMING OF CAB EXPRESSION 1)
• LHY (LATE ELONGATED HYPOCOTYL)
• CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)
• PSEUDO-RESPONSE REGULATOR 7 (PRR7)
• PSEUDO-RESPONSE REGULATOR 9 (PRR9)
► TOC 1 belongs to PRR family (PSEUDO RESPONSE
REGULATOR)
Gardner et al.(2006) 34

35. OUTPUT PATHWAYS
Outputs pathways  that lead to physiological and biochemical
rhythms.
These clock outputs that ultimately provide the advantages in
growth and competition.
Photosynthesis
Leaf movement Hypocotyl elongation
Stomatal movement
Gardner et al.(2006)
35

36. ORGANIZATION OF CIRCADIAN SYSTEMS
a. linear signalling pathway
b. signalling network
(Harmer, 2009)36

37. MOLECULAR BASIS OF CIRCADIAN RHYTHMS
(Harmer, 2009)
Pieces Still to Be Fit Into the Puzzle
37

38. 38

39. Gene Locus ID Function Loss of Function Overexpression
CCA1 At2g46830 Single Myb domain transcription
factor
Short period Arrhythmic
CKB3 At3g60250 Casein kinase II regulatory subunit Not known (gene family) Short period
CRY1 At4g08920 Blue light photoreceptor Long period in blue light Short period in blue light
CRY2 At1g04400 Blue light photoreceptor Long period in blue light Short period in blue light
DET1 At4g10180 Repressor of photomorphogenesis Short period Not known
ELF3 At2g25930 Unknown Arrhythmic in continuous light Long period
ELF4 At2g40080 Unknown Arrhythmic Not known
GI At1g22770 Unknown Short period, low amplitude Short period, low amplitude
LHY At1g01060 Single Myb domain transcription
factor
Short period Arrhythmic
LUX At3g46640 Myb transcription factor Arrhythmic Arrhythmic
PHYA At1g09570 Red light photoreceptor Long period in far-red light Short period in far-red light
PHYB At2g18790 Red light photoreceptor Long period in red light, leading
phase in white light
Short period in red light, lagging
phase in white light
PIF3 At1g09530 Basic helix-loop-helix transcription
factor
Wild type Wild type
PRR3 At5g60100 Pseudo-response regulator Short period Wild type
PRR5 At5g24470 Pseudo-response regulator Short period Low amplitude, long period
PRR7 At5g02810 Pseudo-response regulator Long period Not known
PRR9 At2g46790 Pseudo-response regulator Long period Short period
SRR1 At5g59560 Unknown Leading phase, low amplitude Not known
TIC Gene not yet identified Short period, low amplitude Not known
TOC1 At5g61380 Pseudo-response regulator Short period Arrhythmic
ZTL At5g57360 F-box protein Long period Arrhythmic
39

40. SUPPORTIVE
EVIDENCES
40

41. Atamian et al. (2016)
41

42. Objectives
Solar tracking movements of stem
Interactions between environmental response pathways
and the internal circadian oscillator
East ward orientations impact on pollination
42

43. Materials and method
Material: Sunflower plant (CM523) and dwarf mutant dw2.
Method: By disrupting the normal processes of plants in two ways:
1. Rotating potted plants
2. By tethering plant stems
Statistical analysis
Linear mixed-effect models
Student ‘t’ test
Instrument
FLIR imaging – Forward Looking Infrared Imaging
43

44. Results
1. The circadian clock regulates solar tracking.
A) Nighttime reorientation of
stem and shoot apex
B) Disruption of solar tracking
by daily evening 180° rotation
of experimental plants results
in a 7.5% reduction in biomass
(left) and an 11% reduction in
leaf area (right)
44

45. D:Persistence of rhythmic
movements after transfer from
field to continuous light and
temperature conditions.
C: Changes in orientation
anticipate dawn and dusk
transitions in both fall (left y
axis) and summer (right y axis)
Contin…
Rate of apical
movement
45

46. E: The onset of “eastward” movement in a growth chamber equipped
with four directional lights is consistently phased with lights being
turned off in 24-hour T-cycles (left and right) but is erratic in 30-hour
T-cycles (center).
Contin…
46

47. 2. Solar tracking is driven by opposing growth rhythms on the east and
west sides of stems.
(B) the angle of curvature of the shoot apex
relative to the horizon in control (green) and
gibberellin-deficient dw2 plants (purple). dw2
mutants were treated twice with 2µM of the
gibberellin GA3 (gibberellic acid), with the
last treatment on day 0.
(C) Timing of elongation for east
and west sides of stems of solar
tracking field-grown plants.
(D) Timing of stem elongation of
plants growing vertically in a top-
lit environmental control chamber.
(A) Changes in stem elongation.
control
dw2+ GA3
Higher growth
rates EvsW
35%
47

48. • (E-H) Differential gene expression on the east and west sides of
solar tracking stems assessed by q-RT PCR
Contin…
48

49. 3. Eastward orientation of sunflower heads after anthesis is due to gating
of light responses by the circadian clock and enhances pollinator visits.
(A) Amplitude of solar tracking and
changes in stem growth of mature
plants nearing floral anthesis. Petals
were first observed during day 5
(B) Stem curvature of
juvenile plants entrained
in 16L:8D cycles and then
exposed to unidirectional
blue light for 4 hours at
the indicated times. 49

50. FLIR images of east-facing (E) and west-facing (W) floral disks at
hourly intervals
Contin…
(D) Pollinator visits to east- and west-
facing plants during 45-min intervals
at three times of day.
50

51. (E) Temperature of sunflower
disks with east and west (with
or without supplemental heat)
orientations.
(F) Pollinator visits in the
morning to the inflorescences
with temperatures reported in
(E).
Contin…
5 fold
51

52. CONCLUSIONS
1. Circadian oscillators enhance fitness by coordinating
physiological processes
2. Coordinate regulation of directional growth is due to
environmental response pathways and the circadian oscillator.
3. Enhances heliotropic movement (young), eastward orientation
promotes increased reproductive performance.
Atamian et al. (2016)
52

53. 53

54. • To know the mechanism of clock-enhanced herbivory
resistance
• Jasmonate harmones are critical for plant herbivore
defense
• The plant circadian clock provides physiological advantage
by performing critical role in Arabidopsis defense
Objectives
54

55. Plant material: All Arabidopsis genotypes have the Col-0 genetic
background except for aos, which is in the gl-1 genetic background.
Seed sources:
 Col-0, aos, jar1, lux2 & CCA1-OX
3 week stage plants.
Cabbage semilooper (Trichoplusia ni) 4 days old were used at the
initiation of every experiment.
Phytohormone Measurements: Measurements were carried out in
selected ion-monitoring mode with retention times (JA & SA).
Materials and method
55

56. METHOD
Incubation of
loopers for 72h
24 h constant dark
condition
Subjecting both
loopers & plant for
light entrainment
In phase
(12 h L/D)
Area of
plant tissue
(mm2)
Weight of
looper (mg)
Out of phase
(off set by 12 h)
Photos of
loopers
compaired
56

57. 1. Arabidopsis is more resistant to herbivory when entrained in-phase rather
than out-of-phase with T. ni looper entrainment.
A. Light/dark cycle entrainment
scheme
Photographs of representative plant tissue
remaining from plants entrained in-phase
and out-of-phase with looper entrainment.
Results
57

58. E. Representative loopers
at 72 h postcoincubation.
C. Area of plant tissue remaining
from plants entrained in-phase
(white bars) and out-of-phase
(filled bars) with T. ni entrainment
after 72 h of incubation without
(control) or with T. ni loopers.
D. Looper wet weights.
Contin…
In
phase
Out of
phase
58

59. 2. T. ni feeding is circadian-regulated, with enhanced eating during
subjective day.
(A) 12 h of light/dark
(B) constant dark conditions
Max @ dusk
Min @ dawn
Max @ dusk
Min @ dawn
59

60. 3. Arrhythmic Arabidopsis plants lack enhanced herbivory
resistance when entrained in-phase with T. ni loopers.
A. Plant tissue remaining
from CCA1-OX(transgenic)
and lux2(mutant) entrained in-
phase and out-of-phase with
T. ni entrainment after 72 h of
plant-T. ni coincubation.
B. Area of plant tissue remaining
from plants entrained in-phase and
out-of-phase with T. ni entrainment
after 72 h of incubation without
(control) or with T. ni
60

61. C. Wet weights of T. ni
fed on in-phase and
out-of-phase plants.
D. Representative T. ni loopers
at 72 h postcoincubation.
Contin…
D
These data suggests that Arabidopsis circadian clock is essential for
enhanced plant defence against T.ni herbivory when entrainment is
synchronized. 61

62. 4. Jasmonates are required for enhanced herbivory resistance
Goodspeed et al. (2012)
A. Plant tissue remaining from
gl-1, aos, and jar 1 entrained in
phase with T.ni entrainment
B. Area of plant tissue remaining
from plants entrained in-phase and
out-of-phase with T.ni entrainment
after 72 h of incubation without or
with T.ni
62

63. C. Wet weights of T.ni fed on
in-phase or out-of-phase plants
D. Representative T.ni loopers
E. Jasmonate (20-35%) and salicylate
accumulation patterns are circadian-
regulated with opposite phasing
JA SA
Contin…
63

64. The plant circadian clock provides a strong physiological advantage
by performing a critical role in Arabidopsis defense.
The daily herbivory battle between T. ni and Arabidopsis, evolution
of the circadian clock gives the advantage to the plant.
CONCLUSIONS
64

65. • Molecular techniques
• Infra-red gas exchange Analyzer (IRGA)
• Leaf movement as a circadian reporter
• Transgenic luciferase as a circadian reporter
Techniques for assaying circadian rhythms in plants
65

66. 66

67. 67

68. FUTURE PROSPECTS
• Circadian clocks in abiotic stress responses
• Circadian clocks in plant defense
• Circadian clock in hybrid vigour
68

69. • Helps in regulation of plant growth and development
• Promotes plant fitness by synchronizing endogenous clock with
environmental cues
• Application of circadian clock genes has just been started
exploiting in crop breeding, hence there is a need to breed crops
that can adapt to diverse environment.
CONCLUSION
69

70. BSK
70

71. The circadian clock is an important integrator of environmental
cues that coordinates the physiological response of the plant
through a complex genetic network.
The ability to asses circadian clock function and variation will lead
to significant advances in our understanding of the interactions
between the circadian clock and plant fitness.
Understanding the genetic contributions to changes in flowering
time in response to photoperiod, temperature and precipitation is
critical towards expanding the geographical distribution of crops
as well as their adaptability to the changing environment.
71