Week 6 Flashcards
What environmental conditons vary over 24 hours?
Temperature
Light intensity
Humidity
Predator behaviour
What is the example of extreme day-night temperature change?
57 degrees c
Montana, 1972 -48 to 9
What is a more typical day-night fluctuation?
10 degrees celcius each day (seen in central japan)
What is the mechanism for circadian clocks?
Biological oscillators with a rough 24 hour period
What did they find when the kept mice in constant darkness?
Continous darkness rhythms persist but with a 23 hour period
Most wheel running at night
When was the circadian rhythm first discorvered in plants?
1729 by Jean-Jacques d’Ortous de Mairan in Mimosa pudica
What is the stages of time course analysis?
First plant is grown in cycles of light and dark (this can be drawn in a graph with light and time on x axis and biological process on the y)
What happens to a plant grown in light and dark cycles when moved into continous light?
In constant light and temperature conditions the circadian rhythm will form whats called a ‘free run’
What is ‘free run’?
Circadian rhythm continous but elevated graph ie still running but with a peak and trough
The period that would have been night is subjective night
The period that would have been day is subjective day
What is a period?
Time to complete one full cycle
What is a phase?
The time at which a particular point of cycle occurs (e.g peak)
What is the amplitude?
The displacement of the oscillation from the centre point
What is the advantage of a functioning circadian clock?
Plants with functioning circadian clock grow larger
What plant activities are controlled by the circadian clocks?
In Arabidopsis thaliana around 30% of genes oscillates with a 24 period
Stomatal opening and closing are under the controle of the circadian oscillator
Hypocotyl elongation is clock controlled
Photoperiod is one of the environmental factors controlling flowering
In competition experiments what are the benefits of functioning circadian clock?
Higher:
Survivial
Biomass (dry and fresh rate)
Chlorophyll content
What was the experiement and result of changing the circadian cock period?
toc1-2 (20 hour day) ztl-27 (28 hour day)
toc1-2 was bigger in the 20 hour day compared to other plant and the same plant in the 28 hour day
ztl-27 was bigger in the 28 hour day compared to other plant and the same plant in the 20 hour day
What is the circadian oscillator?
Generate a rhythm with a ~24 hour period within the cell
What is the entrainment pathways?
Synchronise the oscillator with the external time of day so that the clock stays accurate
What is the output pathway?
Communicate temporal information form the oscillator to other parts of the cell
What is the circadian gating?
Adjust the sensitivity of entrainment and output pathways depending on the time of day
What is the pathway of the circadian system of plants?
Envrironemental inputs –> Entrainment pathways –> Circadian oscillator –> Output pathways –> Rhythms in transctiption, physiology and biochemistry
What is the system that oscillates for the circadian clock?
Transcription-translation feedback loop
What is the rough transcription-translation feedback loop?
Protein encoded by Gene A activates Gene B
Protein encoded by Gene B supresses Gene A
What are the key points of a simple biological oscillator?
Reciprocal feedback loop
Negative feedback step
Speed of biochemical reaction adds a rate constant
Who were the scientist who identify the genes forming the circadian clock?
Prof Steve Kay FRS (Uni of Southurn California)
Prof Andrew Millar FRS (Uni of Edinburgh)
What is the first step in identify genes forming circadian clock?
Made a transgenic Arabidopsis expressing a promoter-luciferase reporter for a circadian regulated gene
(CAB2 promoter, chlorphyll A/B binding protein2)
Just need to measure bioluminescence controlled by CAB2 promoter
What is the second step in identify genes forming circadian clock?
Mutagenise enormous number of seeds of CAB2:Luciferase transgenic plants, and screened the mutant population for changes in the circadian rhythm of CAB2:LUCIFERASE
(Some mutations fall within genes forming parts of the circadian clock, so disrupting the rhythm of CAB2:LUCIFERASE)
What was the early model for the functioning of the circadian clock in Arabidopsis?
Oscillator with acitvation with supression feedback
Main genes involved TOC1, LHY and CCA1
Why is the early model for circadian clock in Arabidopsis out of date?
TOC1 supresses CCA1, though this is a good example of oscillator structure
What is reciprocal repression between CCA1 and TOC1 at the core of the circadian clock?
Overexpression of CCA1 supresses circadian oscillation of TOC1 (CCA1 and LHY bind to the promoter of TOC1)
Overexpression of TOC1 supresses circadian oscillation of CCA1 (The CCT domain of TOC1 is required for it to bind the CCA1 promoter)
What are the two major loops in the circadian clocks?
Morning loops and Evening complex
What do Network modles indicate?
Connections between components but lack temporal information about clock function for example 3 genes CCA1, PRR9 and Lux all changing throughout the day
Why is entrainment required?
As dawn and dusk is different everyday
The period of the circadian oscillator is approximately 24h and there is natural variation between plants
How do circadian clocks regulate plant cells by controlling gene expressions?
Some circadian clock proteins are transcription factors that regulate sets of genes with a circadian rhythm
What is an example of clock regulating plant cells?
Daytime transcription factors –> transcription factors are active and genes transcribed during the day but are doesnt happen during the night
How can you analyse the extent of circadian regulation?
Sample RNA over a 24h or 48h period then analyse transcripts from all genes using microarray or RNA sequencing methods
What did early studies find about the importance of the circadian rhythms?
Found 6% of transcipts have circadian rhythms –> many photosynthesis genes have circadian rhythms (e.g photosystem 1 and 2)
What underlines specific circadian phases of transcription?
‘cis elements’ (short, specific sequences)
Where do cis elements appear in high frequency?
They appear frequently in promoters of transcripts which can be used to identify time of day of transcripting at certain circadian phases
What do cis elements do?
Indicates when the circadian clock regulates different circadian phases through particular clock-controlled promoter motifs
How wide spread are circadian controlled genes?
Almost all metabolic pathways include at least one enzyme that is under circadian transcriptional control
When is chlorophyll biosynthesis peak?
Just before dawn to anticipate light avaliability
When does starch catabolism genes peak?
Around dusk
What does the mutant called prr9/7/5 do?
Increase in shikimate suggests changes in secondary metabolism and increase in Citric Acid cycle intermediates
How did scientists find out the impact for the mutant prr/9/7/5?
Metabolomics
How does a plant make sure it doesnt deplete starch reserves before dawn?
The rate of starch degradation is related to the length of night so that the plant only exhausts starch reserves just before the end of the night
What does the cca1/Ihy mutation do?
Causes the plant to exhaust starch reserves at night
What is the impact to the plant with the cca1/Ihy mutation?
Accumulates 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 night
Where does the gene controlling chloroplasts circadian rhythm?
The gene SIG5 is made in the nucleus which is then imported into the chloroplast
What is the overall factors for controlling the circadian rhythm?
Environmental signalling, central oscillator and metabolism all impact each other
What is circadian gating?
The regulaiton of other cell signalling pathways by the circadian clock
What is the importance of circadian gating?
Fundamental way that circadian clocks regulate plant cells
What happens 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 ont he time of day
What is the general principle of light gating?
Light stimulus –> gate open –> strong response to light
No light –> gate closed –> weak response to light
Why does the circadian clock regulate its own sensitivity to light?
The way it responds to light depends on the time of day through entrainment
What happens if the circadian clock responds to light every time of day?
It would be rest to dawn continuously and unable to to provide a measure of time
How does the circadian clock gate respond to a cold environment?
Nonfreezing cold temps cause plant to prep
Plant response during subjective day compared to subjective night (may not be accurate due to always under light and given suddencold shock treatments)
What does circadian gating impact?
Entrainement pathways and output pathways
How does Ppd-H1 impact photoperiodic responses of barley?
Responds to day lengths in excess of minimum to flower in long-day plants but not present in other varieties
How was Ppd-H1 discovered in Barley?
Breeding photoperiod sensitive ‘Igri’ with photoperiod insensitive ‘Triumph’ –> genetic mapping of population generated –> Ppd-H1 locus indentified
What did they find about the Ppd-H1 gene in Barley?
It is a homologue of the Arabidopsis PRR7 pseudo-response oscillator gene (around 50% similarity)
What is skotomorphogenesis?
The development of a plant in darkness
What are the key features of skotomorphogenic plant?
Long hypocotyl
Incapable of photosynthesis
Closed unexpected cotyledones
Apical hook
Etiolated (decoloured due to lack of light)
Adapted to underground growth
What is photomorphogenesis?
The process of plant development in light
What are the key features of photomorphogenic plant?
Shorter hypocotyl
Capable of photosynthesis
Expanded cotyledons
Adapted to growth in light
What changes a plant from skotomorphogenic to photomorphogenic?
Red light
What happens when a seed is exposed to red light then soon after far red light?
The seed will stop germinating
What happens when a seed is exposed to red light then soon after far red light followed by red light?
The seed will germinate
What processes the red/far red reversibility?
Phytochromes
What does red light do to plants at different developmental stages?
Seed - Promotes germination
Seedling (skotomorphogenic) - Promotes photomorphogenesis
Seedling - Promotes formation of leaf primordia
Adult - Inhibits internode elongation
Adult - Inhibits flowering
What is the hypocotyl?
The part of the stem of an embryo plant beneath the stalks of the seed leaves or cotyledons and directly above the root
How big is the phytochrome protein?
125 kDa
What are the 2 forms of phytochrome?
Pr and Pfr
What converts Pr to Pfr?
Red light
What converts Pfr to Pr?
Far red light
What is the the colour and light absorbed by Pr?
Colour: Blue
Absorbs red light
What is the the colour and light absorbed by Pfr?
Colour: Blue-green
Absorbs far-red light
What is the percentage of Pr:Pfr in red light conditions?
15% Pr: 85% Pfr
What is the percentage of Pr:Pfr in far red light conditions?
97% Pr: 3% Pfr
What do both Pr and Pfr both absorbs?
Both absorb blue and UV-A
What is the max wavelength absorbed by Pr?
666 nm
What is the max wavelength absorbed Pfr?
730 nm
What are the 2 potential causes for Pf and Pfr ratio change in red light?
Either decrease in Pr or increase in Pfr
What evidence that Pfr accumulation induces a response?
Magnitude of response is proportional to amount of Pfr
In Arabidopsis what do hy proteins do?
HY1 and HY2 are required for chromophore biosynthesis
In the experiment with hy mutants what does the change in light do?
In wildtype there is a reponse in plant when in red light when was in dark
In hy mutant there is a lack of a response
Why is there a lack of a response in hy mutants?
As HY1 and HY2 are required for chromophore biosynthesis and hy1 and hy2 mutants lack active phytochrome, thus no response
What is the non-protein part of the phytochrome?
The chromophore
What are the 2 halfs of the protein part of the phytochrome?
N-terminal domain
C-terminal domain
Which half of the protein is the chromophore attachted to?
Half way through the N-terminal domain
What is halfway through the protein part of the phytochrome?
the hinge region
What is the structure name for the whole cytochrome?
Holoprotein
What makes up a holoprotein?
Apoprotein + chromophore
What is the size of phytochrome when purfied from plants?
250 kDa, two phytochromes stuck together
What is the structure name of the chromophore?
Phytochromobilin
What joins the chromophore to the protein?
Thioester linkage between Cys and the sulphur in the chromophore
What is the structure of chromophore?
Covalently linked linear tetra-pyrrole
Where is chromophore produced?
In the plastid and then diffuses into the cytosol
What is the difference in structure between Pr and Pfr?
Pf = Cis
Pfr = Trans
What does red light do to the structure of chromophore?
Changes the structure in chromophore which causes structural change in protein
How many phytochrome genes does Arabidopsis have?
5
Which phytochrome gene in Arabidopsis is type 1?
PHYA
Which phytochrome gene in Arabidopsis is type 2?
PHYB
PHYC
PHYD
PHYE
Which phytochrome genes do monocots have?
PHYA, PHYB and PHYC
What is the difference between PHYA and phyA?
PHYA = gene/ apoprotein
phyA = mutant allele/ holoprotein
What is the difference between PrA and PfrA?
Pr of PHYA
Pfr of PHYA
What is fluence?
Number of photons/unit area
What is fluenced meausured in?
mol_m^-2
What is fluenced rate?
No photons/unit area/unit time
What is fluenced rate measured in?
mol_m^-2s^-1
What is the fluence formula?
Fluence = fluence rate x time
What is VLFR?
Very low fluence response
What is LFR?
Low fluence response
What is HIR?
High irradiance response
What is an example of VLFR in dicots?
Seed germination
What is an example of LFR in dicots?
Hypocotyl elongation (PHYC), petiole elongaiton (PHYD)
What is an example of HFR in dicots?
HYpocotyl elongation (PHYA) and Flowering (PHYB)
What is an example of VLFR in monocots?
Coleoptile elongation (PHYA)
What is an example of LFR in monocots?
Mesocotyl elonagation (PHYB)
What is an example of HFR in monocots?
Brace root gravitropism (PHYA)
What are phyA mutants?
Plants that are compromised in far-red light induced photomorphogenesis
What are phyB mutants?
Plants that are compromised in red light induced photomorphogenesis
What happens to phyA plants grown in red and far red light?
Red = photomorphogenetic
Far-red = not photomorphogenetic
What happens to phyB plants grown in red and far red light?
Red = not photomorphogenetic
Far-red = photomorphogenetic
What is the ratio of red to far red light in sunlight?
Red 1.19: 1 Far red
What is the ratio of red to far red light at ivy canopy?
Red 0.13: 1 Far red
Why is there a drop in red to red light ratio as you go down a canopy?
Due to red light being absorbed by plants
How do sun plants responsed to less red light in plants?
This causes them to extend their length
What is the difference in protein structure in Pr to Pfr
N-terminal domain (photo-sensing domain) is closely associated to the nuclear localisation domain in Pr. In Pfr they are less closely associated with a more open structure, exposing nuclear localisation signals
Where is phyA-GFP and phyB-GFP when its dark?
Spread around the cell
Where is phyA-GFP and phyB-GFP when its light?
Localised in the nucleaus
What happens to Pfr when is it is formed from Pr?
It enters the nucleus
What are the two functions of Pfr in the nucleus?
Inhibits the formation and degrades currently made PIPs and PILs
Inhibits degradation of light response transcription factors
What do PIPs and PILs do?
They inhibite light responses and are always made in the dark
How does Pfr degrade PIPs and PILs?
Binds to E3 Ub ligase which attaches ubiquitin, then they are targeted to the ptoteasomes and broken down
What happens light transcription factors during the night?
They are broken down
What are examples of light transcription factors?
HFR1, HY5 and LAF1
