Circadian rhythm

circadian rhythm in plants

1. CIRCADIAN RHYTHM IN PLANTS 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 (clock Δ19) 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 …??? (Harmer, 2009) 20

21. ( 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. 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). 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 ( Somers, 1999 ) 24

25. 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 - Q 10 value will be nearly 2 - 3 (approximately double the rate of the process ) In contrast, the Q 10 values of circadian rhythms lie between 0.8 and 1.4 ( Q 10 = 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. C locks 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

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

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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- , 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 56

57. 1. Arabidopsis is more resistant to herbivory when entrained in-phase rather than out-of-phase with T. ni looper entrainment. 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 … 1.6 5 .16 In phase Out of phase 58

59. 2. T . ni feeding is circadian-regulated, with enhanced eating during subjective day. 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 c oincubation . 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

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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