Circadian Rhythms

Question 1

early recordings of c rhythms

Answer 1

linneus - noticed different flower sp opened at diff times of day.
De Marian - 1729. mimosa pudica folds up leaves at sunset

Question 2

what is and what causes c rhythms?

Answer 2

biological oscillators within cells within 24h.

due to rotation of earth causing variation in temp, light intensity, humidity and pred behaviour.

Question 3

what do c rhythms control

Answer 3

physiology - stom conductance -> CO2 assimilation and gas exchange
growth - hypocotyl extension
molecular biology - 30% of arabidopsis transcriptome oscillated within 24h.
photoperiod - eg WT arabidopsis is small and flowers in log days, whereas an arhythmic mutant is large and not flowering.

Question 4

when does a c rhythm give a fitness advantage?

Answer 4

when internal clock matched environment, plants perform better in experiments, in terms of biomass, survival and chl content.

Question 5

what data shows that a c rhythm matching the environment will grow better

Answer 5

comparison of WT and CCA1-OX.
grown in T20, T24, T28
T24 most growth, almost double T20 and T28.
CCA1-OX accumulated less biomass.

Question 6

what is time course analysis?

Answer 6

plants grown in cycles of light and dark, monitor progress.

if transferred to constant light/dark, can see free-run of c regulated bio processes.

Question 7

4 components in a circadian system

Answer 7

1. C oscillator - generated rhythm over 24 h period in the cell
2. Entrainment pathways - synchronise oscillator with external time so clock stays accurate
3. output pathway - communicates temporal info from oscillator w other parts of the cell
4. c. gating - adjusts sensitivity of entrainment and output pathways depending on time of day.

Question 8

how does a c oscillator work?

Answer 8

reciprocal feedback loop of transcription/translation

eg gene A encodes transcription factor of B. B encodes inhibitor of A

Question 9

describe the arabidopsis early model of the oscillator

Answer 9

TOC1 gene - encodes TOC1 protein
TOC1 protein upregulates LHY/CCA1.
if LHY/CCA1 is OE, inhibits TOC1 gene.

the current model is a v complex network of morning and evening complex.

Question 10

4 post transcriptional processes involved in c clock, eg of how theyrelate to arabidopsis.

Answer 10

1. chromatin remodelling - TOC1 promoter clock controlls H3 acylation, which affects TOC1 expression.
2. control of protein stability by protasome. eg ZTL in dark dependent degredation of TOC1 protein.
3. phosphorylation - CK2, casein kinase 2, phosphorylates LHY/CCA1
4. cytosolic signalling molecules. C rhythms of CADPR and Ca2+ in cytosol regulate dynamics of oscillator.

Question 11

what is entrainment and how does it work, breifly

Answer 11

matching of internal rhythm to external time, as the timeof dawn and dusk is different every day.
invovles phytochromes, cryptochromes, tempinfo and sugars from photosynthesis.

Question 12

experiment of entrainment

Answer 12

testing the ‘sugar pulse effect’ on arabidopsis c clock
adding sugar at different times causes shift in c clock.
Changes in clock gene expression
CCA1
add sugar in morning causes a phase shift and alteration of CCA1 expression. requires PRR7 gene (oscillator gene)

Question 13

describe c control of carbohydrate degradation

Answer 13

carbohydrate degradation
temporarily controlled at night. rate of degradation related to length of night so only exhausts reserves just before dawn. changes rate is unexpected start of the night.
CCA1/LHY mutants exhausted starch reserves. accumulated 20% less than WT, and degrades 35% faster at night.
exhaust reserve 3-4h earlier and express starvation genes.

Question 14

describe c control of metabolic pathways

Answer 14

transcriptome analysis has shown many ph genes in arabidopsis have c rhythm.
many c clock proteins are TFs.
usually >1 enzyme in all metabolic pathways is under c control.
eg. chl synthesis- gene transcripts peak before dawn. Starch catabolism genes peak around dusk.
c clock mutants have diff metabolite levels in light/dark cycles.

Question 15

what is circadian gating

Answer 15

regulation of cell pathways by c clock.
acts a a ‘valve’ on plant response.
same environmental cues have diff strength responses.
if gate is open, strong response, eg in day. at night gate might be closed, so weak response.

Question 16

2possible ways c gating works

Answer 16

1. gate signals entrain the clock - as if clock responded identically to light all the time, would be reset to dawn continuously. regulates own sensitivity to light, so response depends on time of day.
2. ## signalling pathways regulate response

Question 17

3 examples of gating

Answer 17

1. day - more sensitive to cold than night. seen from examining gating of CBF gene at diff times of day.
2. gating of shade av response, examines hypocotyl elongation. more elongation at specific times of day.
3. sensitivity to pathogen flagellin proteins changes across 24h. the time of day which bacteria arrives elicits different strength response.

Question 18

how are chloroplast genes controlled with a c rhythm?

Answer 18

chloroplast gene exp controlled by c rhythm of nuclear genes.
eg SIG5 gene protein if imported into the chloroplast where it binds to chloroplast genes and acts as a TF.

Question 19

give an example of a crop in which only one gene would need to be altered to change c rhythm

Answer 19

barley - long day plant, has a minimum day length to induce flowering. some varieties are insensitive to day length. many genes homologous to arabidopsis circadian genes. eg PpdH1 homologue of PRR7, oscillator gene.
PpdH1 is day length sensitive allele
ppdH1 is insensitive allele. Spring barley has this allele and flowers in winter. Winter barley only flowers in early spring. 1 gene difference.

Question 20

why is seasonality important?

Answer 20

controls plant dev
arabidopsis - short day - bushy, no flowers, large rosette.
long day - spindly with flowers.
plants detect season by comparing the internal cycle with external conditions.

Question 21

3 groups of seasonal plants

Answer 21

1. long day - arabidopsis
2. short day - rice, soy
3. day length neutral - flowering is insensitive to photoperiod.

Question 22

how can flowering be induced in a longday plant in short days?

Answer 22

long day plant doesnt flower if nights are long.

flash of light at night will induce flowering.

Question 23

genetic control of flowering

Answer 23

FT locus - FT protein is expressed in leaves, moves to meristem through phloem to initiate flowering organs. induces floral integrator genes.
FLC represses FT. upstream of FT.
CONSTANS gene required for floral induction in long days.

Question 24

CONSTANS

Answer 24

CO is a zing finger TF, circadian dependent, broadly conserved throughout plants.

dark - CO protein unstabledue to COP1 activity. doesn’t accumulate even though CO mRNA does.
light: CO protein and mRNA accumulate as no COP1. CO induces FT. (opposite day and night in rice)

Mutant: CO-
looklike short day plant in long days. doesnt flower.

Question 25

describe levels of CO protein, CO mRNA and FT mRNA in light and dark, in long day and short day rice and arabidopsis

Answer 25

see graphs

Question 26

what is vernalisation?

detailed pathway of how it accelerates flowering

Answer 26

Silencing of FLC after prolonged period of cold by histone modification H3k27Me3.

caused by PRC2 complex. polycomb repressive complex 2. PRC2 is always on histones, but after vernalisation found VRN2, VRN5 and VIN modifications spread along histones.
COOLAIR - antisense RNA produced at FLC locus after cold. required for another epigenetic switch at FLC., reduces H3k27Me3.

Question 27

long term seasonal temp trends of FLC

Answer 27

2-3weeks cold - FLC decreased to the lowest level.
4-6 weeks cold - FLC suppressed stably after cold period.
seasonal change in FL expression - winter: none. Spring increases. Summer: highest.
FLc responds to temp over past 42 days.