M1: Signalling Flashcards
Describe the concentration difference of Ca2+ across the plant cell
- Lower conc (nM) in the cytoplasm
- Higher conc (mM) in the apoplast, vacuole and ER
- Self regulation of Ca2+ concentrations occur in each organelle
What are the different ways to measure [Ca2+]? (4)
- Luminescence Indicators (Aequorin)
- Electrode (patch clamp)
- Chemical fluorescent Calcium Dyes (Fura-2)
- Genetically Encoded Calcium Indicators (CAMELEONs, GCaMP)
Describe the function of aequorin as a Ca2+ indicator
- Apoaequorin transformed into cells
- Binds to coeleterazine to become the active form aequorin
- Can bind up to 3 x Ca2+ in the EF hands
- Binding leads to oxidation reaction emitting blue light
= Intensity is proportional to the conc of Ca2+
What are the positives and negatives of using aequorin?
Positives
- Non-invasive
- Wide calcium detection range
- Quantitative measure
- Low background signal
- Low phototoxicity, doesn’t require external excitation
Negatives
- Irreversible (use once)
- Lower sensititivity
What are the adv and disadvantages of using electrophysiology to measure [Ca2+]?
Adv
- Direct measurements of Ca currents are taken
Disadv
- Difficult to accurately place the electrodes in the cell
- Takes time to reach resting potentials
- Invasive and disruptive
Describe the function of Ca2+ indicators
- Derived from Ca2+ chelators that bind to Ca2+
- Can be chemical (Fura-2) or genetically encoded (GCaMP, GFP, CAMELEONs)
- Upon binding to Ca2+ the structures undergo an optical change and emit different fluorescence
What are advantages and disadvantages of calcium indicators?
Adv
- Real time monitoring
- High sensitivity
- Non- invasive
- Versatile
Disadvantages
- Phototoxicity + Photobleaching
- Background noise
- Interference with functions e.g. high conc of Ca2+ buffer will reduce signal strength etc.
Compare Calcium Green and Fura-2
- Number of peaks formed
Calcium green (1), Fura-2 (2) - Measurements
Fura-2 requires rapid flipping between two filters to measure intensity then carry out a ratio metric analysis = MORE ACCURATE/ QUANTITATIVE
Calcium green fluorescence levels is then directly used to analyse concentrations = QUALITATIVE
Describe the structure and function of CAMELEONs
CFP - cyan fluorescent protein
YFP - yellow fluorescent protein
CaM - calmodulin (binds to Ca2+)
M13 - peptide that binds to CaM in the presence of Ca2+
- Presence of calcium leads to binding of CaM and M13, bringing YFP closer to CFP leading to different wavelengths being released
Describe the structure and function of GCaMP
- GFP-based calcium indicator
- enhanced GFP (eGFP) encoded backwards
- CaM and M13 present
In the presence of Ca2+ M13 binds to CaM causing changes in the gene structure and allows for coding of EGFP, which then emits fluorescence - Not ratiometric, however, lack of fluorescence indicates lack of Ca2+
What are the 6 different plant hormones and their roles?
Auxin - cell elongation, apical dominance
Gibberellins - stem elongation
Cytokinins - cell division, leaf senescence
Brassinosteroids - cell elongation and division
ABA - stress response
Strigolactones - microbial signalling
What is cross-talk?
Overlap between two different signals, that converge at a node and generate an integrated response
What is the meaning of ectopic expression?
Expression of a gene where it isn’t normally found e.g. not in roots or shoots
Or expression in a different species
- Used to show similarities and function of the gene
What are the characteristics of signalling networks?
- Appropriately sensitive to the stimulus
- Specific to stimulus
- Propagating signal
- Robust
- Refractory (switch on and off)
How do proteins transfer information?
- Conformational change
- change in shape leads to downstream signalling e.g. kinases - Covalent modification
- Transfer of functional group e.g. phosphate via kinases and phosphatases
What are the characteristics of the components (receptors etc.) in the signalling pathway?
- Position
- Sensitivity (Kd)
- Specificity
- Capacity to relay
- Timing
Give an example of cross-talk and sharing of co-factors
- FLS2 shares the co-factor BAK1, with BRI1
- Involved with brassinosteroid signalling
- Epidermal kymograph shows lack of clustering, even though they have the same co-factor
What is the scaffold hypothesis?
- Tether signalling components from various pathways
- Co-ordinates +ve and -ve feedback signals
- Insulating correct signalling proteins preventing inactivation
e.g. FERONIA that brings FLS2 and EFR to meet BAK1
Give examples of positive and negative feedback loops with FLS2
Positive
- flg22 binds and activates FLS2 which activates WRKY22/29 to activate their own production
- FLS2 initiates MAPK pathway, increasing SA which enhances expression of FLS2
Negative
- Activation of negative regulatoes, targeting FLS2 for ubiquitination leading to endocytosis. Preventing excessive energy expediture or autoimmunity
- Or ABA increasing NO production which then deactivate ABA receptors
What are the causes for [Ca2+] cyt increase?
- Abiotic stress
- Biotic stress
- Symbiotic signals
- Hormones
- Signalling intermediates (ROS, NO, cAMP)
What experiments can be done to find out where Ca2+ is derived from?
- Blocking channels
- Lanthanum ions, similar radius to Ca2+ can bind and block - Chelating Ca2+
- EGTA to Ca2+ to prevent movement - Calcium channel mutants
- knock out CNGCs (mutation in CNGC11/12 activates defence responses) or TPC1 (high extracellular [Ca2+] led to stomatal closure in WT and overexpressor, but the tpc1 mutant was unresponsive. (Peiter et al., 2005))
How can specificity be generated in Ca2+ signals?
- Cell specific promoters/ transporters
- Monophasic/Biphasic/ Oscillatory signals
- Amplitude of the signal
- Duration and frequency
What Ca2+ changes occur in chloroplasts?
- Thylakoids a greater store of Ca2+ compared to the stroma
- Changes in Ca2+ in response to light (circadian) controlling photosynthesis
– Aequorin based experiment showed that stroma Ca2+ responds differently to same stress
What evidence is there that nuclear Ca2+ changes differ to cytosolic Ca2+ changes?
Addition of oomycete PAMP (cryptogein) leads to slower increase in Ca2+ in nucleus, compared to the cytosol
- Nuclear Ca2+ oscillations can occur independent of cytosolic oscillations, indicating distinction between the two
- Another reasoning for differences, is that the nucleus Ca2+ changes have an effect on gene expression, while cytosolic changes have an effect on ion movements
How can Ca2+ channels be located/ discovered?
- Patch clamp electrophysiology
- Discovery of TPC1 - Planar lipid bilayers
- vesicles bind to artificial membrane and measure changes - Genomics looking at homology
- Discovery of GLRs and CNGCs - Genomics looking at mutant screens
What does TPC1 encode?
Slow vacuolar channel
- Activated by increasing [Ca2+] in cytosol
- 2 pore regions, forms a dimer
- Located on the vacuole
- High extracellular [Ca2+] in guard cells, shows greater stomatal closure in WT and overexpressors, however, tpc1 mutant is unresponsive (Peiter et al., 2005)
Name 2 types of Ca2+ channels?
- Glutamate Receptor-like family (GLRs)
- 1 pore region, suggests tetramer formation
- Encoded in the nucleus, but expressed in the thylakoid and mitochondria
- Bind to a range of amino acids - Cyclic Nucleotide Gated Channels (CNGCs)
- Subunits only contain 1 pore region, suggests tetramer formation
- Found on various membranes, leads to greater diversity
- Mutation in CNGC11/12 activates defence response and enhanced resistance (McAinsh and Pitman, 2009)
What are 2 types of mechanosensitive channels?
- Mid1 Complementing Activity (MCA1)
- No pore region present
- Mutants showed reduced response to cold stress in cytosolic aequorin experiments. Double mutant MCA1/2 shows no difference suggesting same pathway - OSCA1 = Ca2+ channel gated by osmotic pressure
- One pore region, forms tetramer
- Localises to plasma membrane (GFP experiment)
- Mutant shows reduced Ca2+ mobilisation, in response to changes in osmolality
- ABA induced stomatal closure was no different in osca1 mutants, and no difference seen in response to H2O2 which causes [Ca2+] suggesting specificity to osmolality (Yuan et al., 2014)
What are annexins?
Soluble proteins that can bind to Ca2+ and localise to a membrane facilitating transport
What are two types of channels that sequester Ca2+?
ECA - endomembrane, found on golgi or ER. Help maintain low cytosolic Ca2+
ACA - found on plasma membrane, autoinhibited require activation by CaM. Pumps Ca2+ out of the cytosol
Name an example other than ACA of a transporter that is autoinhibited at the N terminus
CAX1
- low affinity antiporter of Ca2+/H+ across the vacuole membrane
- autoinhibited by Cax interacting proteins instead of CaM
What would be the result of CAM7 OE on COP1 and HY5? Phenotypically how would the plant present?
Phenotype = photomorphogenic (short hypocotyl)
- OE leads to greater COP1 suppression, and more HY5
- CAM7 is a positive regulator of HY5, by binding to the promoter region
What is the function of CAM7?
Active in light conditions, promoting the action of HY5 in inhibiting hypocotyl elongation
- Also present in the dark, however, unable to overcome effect of COP1 ubiquitination of HY5
What are the 4 key Ca2+ decoder families?
- Calmodulin (CaM)
- Calmodulin-like (CML)
- Ca2+ dependent protein kinase (CDPK)
- Calcineurin B-like (CBL)
Describe the structure and target of calmodulin
Structure - 2x EF hands, binding 2x Ca2+ each
- Located in the cytosol, nucleus, chloroplast and cell wall
Target (transporters, TFs, kinases and enzymes)
- Switches off CNGCs
- Switches on ACAs
- MK phosphatases as part of MAPK cascade
- CAMTA3 (a TF) when Ca2+ high CaM binds to CAMTA3 and suppresses transcription of immune related genes
Describe the structure and target of CAM-like proteins (CML)
1-6 EF hands
Response - to mechanosensing in roots, cml24 mutant unable to respond to mechanosensitive stress
Targets (transporters, kinases)
- CML36 targets ACA8 (Ca2+ ATPase involved in sucrose signalling)
- Targets protein kinases and transcription factors
Describe the structure and target of CDPKs
- Kinase domain and a CAM-like Ca2+ binding domain (EF hands)
Targets (transporters, enzymes, TFs) - ACA8
- SLAC1 by CPK6 and 21
- form part of decoding of flg22 pathway
- CPK8 negatively regulates BIK1 by Pi and ubiquitination
Describe the function of CBL and CIPK and their targets
- work together, CBL interacts with CBL interacting protein kinase (CIPK)
Targets (transporters only) - transporters such as SOS1 (Na+/H+ antiporter)
- Activation of AKT1, specifically CIPK23/CBL1/9 in K+ deprivation
Explain the importance of the interaction between CNGC15 and DMI1
Important for the establishment of plant-microbe interactions
- DMI1nuclear membrane localised ion channel regulating release of Ca2+
- CNGC15 also located on the nuclear membrane and helps sustain Ca2+ flow generating Ca2+ spike and oscillations (important for re-uptake)
What CNCGs are involved in root gravitropism? Explain their function
CNGC10 - mutant had lower Ca2+ levels and unable to respond to gravitropism
CNGC14 - mutant shows impaired response to auxin
= Ca2+ influx important to inhibit elongation
What is required for polar growth of the root hair? What genes are involved?
- Ca2+ GRADIENT required, evidenced by conc measurements showing high in the apical and less sub-apically
- CNGC14 (Ca2+ into cytosol, GUS shows greater localisation)
- ACA7 (autoinhibited Ca2+ ATPase) pumps Ca2+ out of cytoplasm, important for resetting (maybe not for gradient)
- KAT1 - for movement of K+ to retain turgor and drive polar growth
What is the role of ACA13?
- Calcium pump regulating calcium homeostasis
- Pump Ca2+ out of cytoplasm into the organelles such as the ER and vacuole
- Ensures that calcium oscillations are reset
POLAR tip growth and pollen tube elongation
What are HACCs, what are their roles and some examples?
Hyperpolarisation activated Calcium Channels
= Via K+ efflux and activation of H+ ATPase, leading to more negative in the cytoplasm
- DMI1, GLRs and TPC1
- CNGCs (mutant analysis found lack of Ca2+ oscillations and abnormal phenotypes)
What evidence is there that homo and heterodimers form with CNGCs?
- Fluorescence labelling, found 4 steps of fluorescence quenching
- Repeat but with two different dyes, found two steps of quenching
What promotes CNGC18 activity?
- Phosphorylation by CPK32
- Regulation by heteromer formation with CNGC7/8
- Activation by Calmodulin, promoting heteromeric channels
- Ca2+ presence
How do AtCNGC7 and AtCNGC8 regulate CNGC18 channel activity?
CNGC7/8 considered silent subunits
- Don’t form channels on their own but regulate other channels
- Co-expression with CNGC18 leads to loss of channel activity
- Regulation by C-terminal interaction
What evidence suggests that CNGC18 predominantly interacts with CNGC8 rather than CNGC7?
Kd (dissociation constants) differ
Lower indication stronger interactions
- Analysis suggest CNGC8 stronger and more stable interaction
How does CaM (calmodulin) regulate the activity of AtCNGC8/18 and AtCNGC7/18 channel complexes?
- CaM acts as calcium binding protein that regulates channel activity
- Regulation by direct interaction depending on Ca2+ conc
- CaM has no effect on homomeric CNGC18 channel
- regulates activity of heteromeric AtCNGC7/18 and AtCNGC8/18
Why is the abundance of CaM2 in pollen significant?
CaM2 abundant in pollen
- specifically activating CNGC18/7 and CNGC18/8
- regulating calcium signatures to control polarised growth
- control of the GLR channel AtGLR1.2
Describe the interplay between reactive oxygen species (ROS) and Ca2+
- RBOH (respiratory burst oxidase homolog) encodes NADPH oxidase (NOX)
- forms radicals and is regulated by Ca2+ binding to EF hands
- mutant rboh can’t form true root hairs, due to lack of Ca2+ gradient
- ROS helps activate Ca2+ channels, generating a +ve feedback loop
Describe the role of ABA in inducing stomatal closure
- Binds to PYL receptors
- Inhibits PP2C and activates SnRK2/OST1
- Phosphorylates RBOH, SLAC1, KAT1, H+ ATPase and HACCs
- HACC leads to [Ca2+]cyt changes
- Targetting CNGC 5,6,9,12 - Activation of K+ efflux channel due to depolarisation
- Loss in turgor of guard cell so stomatal closure
Describe the immune response to flg22
- FLS2 (a PRR) binds to flg22
- Undergoes conformational change and binds to co-receptor BAK1
- Phosphorylation occurs, leading to activation of BIK1
- BIK1 phosphorylates many targets, one of them being OSCA1.3 to promote Ca2+ channel opening. Also primes CNGC4 for activation
- Promoting stomatal closure to prevent pathogen entry
What controls the release of calcium out of the vacuole into the cytoplasm?
- Two pore channel 1 (TPC1)
- voltage gated and activated by Ca2+ increase by Ca2+ induce Ca2+ release, however high is then inhibitory. Present in ABA induced stomatal closure - CNGCs
- gated by cAMP and cGMP - IP3 and InsP6 pathway
- regulating calcium channels
What is the role of CDPKs in ABA regulated stomatal closure?
- Detect increase in [Ca2+] in the cytosol as a result of ABA
1. Phosphorylation of NADPH oxidase to produce ROS
2. Activation of SLAC1 - efflux of Cl- and NO3-
3. Inactivation of inward K+ channels such as KAT1
Describe the wounding response
- Release of eATP
- eATP perceived by DORN1 that controls MAPK and Ca2+ response
- Results in a Ca2+ influx, requires CNGC2 (but no CNGC4), from mutant analysis
- Indirect activation by the production of cyclic nucleotides e.g. cAMP - Initial influx of Ca2+ through CNGC2 leads to activation of Ca2+ induced Ca2+ release e.g. activation TPC1
Describe the calcium response to herbivory
- Detection by PRR and co-receptor binding e.g. BAK1
- Signal tranduction by GLRs (Ca2+ channels) and CNGCs
- Release from intracellular stores (TPC1)
- Downstream targets includes the production of ROS, hormone production and the MAPK cascade activation
Describe the process of initial salt sensing which results in a Ca2+ increase
Due to MOCA1 encoding GIPC sphingolipid
- mutants show reduced Ca2+ oscillations
- MOCA1 is found in the golgi by mCherry experiment
= Important protein in the synthesis of GIPC sphingolipids which moves to the membrane and regulates the structural integrity and charges.
-GIPC negatively charged so can bind Na+, depolarise surface and cause gated channel opening to allow Ca2+ entry into the cytosol
Describe the Salt-Overly Sensitive (SOS) pathway
Dose dependent increase in Ca2+ in response to conc of salt
1. Increase in Na+ in cell leads to Ca2+ increase
2. Ca2+ increase is detected by SOS3 (a CBL)
3. Binds to Ca2+ and undergoes conformational change, allowing it to bind to CIPK (SOS2)
4. Together can phosphorylate targets
5. Promoting efflux of Na+ by SOS1 (Ca2+/H+) antiporter
Describe the initiation, propagation and result of the Ca2+ wave in response to salt stress
Initiation = by MOCA1 and GIPC synthesis
Propagation = by TPC1 triggered by initial Ca2+ entry
Result = Ca2+ leads to activation CPK3 that Pi TPK1, causing K+ efflux and depolarisation, activating TPC1 as a positive feedback loop
What is the role of ROS in creating Ca2+ oscillations?
- Acts as a secondary messenger
- Oxidising cysteine regulators on upstream regulators of CNGCs, controlling their expression
- Oxidise cysteine in TPC1 leading to conformational change
= Promoting positive feedback loops, with increase in Ca2+ increasing activity of NOX, increasing ROS production
Describe the two pathways of ABA induced stomatal closure via the Ca2+ dependent and independent pathway
Independent pathway - ABA binds PYR, activates OST1 to phosphorylate anion channels leading to efflux of Cl- and malate via SLAC1
Dependent - ABA detected by PYR, SnRK2 activated and Pi CNGCs (priming)
What is the role of ABA in stomatal closure, in terms of electrophysiology?
Depolarise the membrane away from -120mV to 0mV
Inhibit the H+ proton pump and K+ influx channel, promoting K+ efflux
How was the PYL receptor to which ABA binds to discovered?
- Screening to find chemical that acts in a similar way to ABA (inhibit seed growth) and found Pyrabactin
- Mutant analysis looking for the receptor
- Found that quadruple mutant for the receptor was unable to respond to ABA
What regulator did ABA insensitive (ABI) plants not encode and what are the downstream effects?
Mutant in PP2C (type 2 protein phosphatase)
- Meant that SnRK activation is constant, so constantly activating Ca2+ influx
= Stomatal closure due to signalling of stress and this can have an effect on development
- Upon addition of ABA show no changes to Ca2+ or ROS production
What is the evidence for ABI/PP2C and PYL interactions?
- In vitro binding assays
- Co-immunoprecipitation assays
- Bi-fluorescence complentation using YFP
What is the role of and mode of action of RGLG1?
Is an E3 Ub ligase degrades PP2C in the presence of ABA (downstream of PYR detection)
- In no ABA RGLG1 is at the plasma membrane
- In ABA RGLG1 moves to the nucleus to degrade PP2Cs
= promoting stomatal closure
Describe the role of OST1 in ABA mediated stomatal closure
- ABA binding to PYR removes suppressive activity of PP2C on OST1
- Autophosphorylation of OST1 occurs
- Activation/ Priming of downstream targets to increase Ca2+ concs to induce stomatal closure
e.g. RBOH, SLAC1
Describe the inhibition of KAT1 in stomatal closure
- Activated at voltages more negative than -120mV (but depolarisation occurs instead)
- Ca2+ increase activates CPK10 to phosphorylate KAT1
- SnRK2.6/ OST1 Pi KAT1 to inhibit also
Describe the activation of GORK1
Efflux of K+ from guard cells
Activated by voltage not Ca2+
- at voltages more positive than around -50 (+50mV from resting)
Reversal potential (no net flow of ions)
- 90mV, close to equilibrium potential of K+ , so more negative means channels are closed
Describe the features of SLAC1
- Efflux of Cl- and Malate
- Activated by ABA, slow
- Active over a wide range of voltages (voltage insensitive)
Activated by Pi by SnRK or CDPK, ABA -> Increase in Ca2+
What changes occur to tonoplast channels in ABA signalling for stomatal closure?
- Ca2+ leads to TPC1 activation, moves out K+ and Ca2+
- TPK1 activated by increased Ca2+, moving out more K+
Describe the process of experimental reconstruction of the ABA signalling pathway
- In xenopus egg
1. ABA responsive element with ABRE binding factor (ABF), isn’t enough for activation
2. Introduction of SnRK to phosphorylate ABF2 did activated ABRE transcribing luciferase
3. Addition of ABI/ PP2C with no ABA, led to inhibition of SnRK
4. Addition of PYR and ABA, inhibited the function of ABI1/PP2C
What is the use of in silico reconstruction for biological pathways?
- See if understanding is correct, by regenerating
- Find new levels of regulation
- Optimising pathways, find rate-limiting steps
What is a monostable and bistable dynamic?
Monostable - one stable steady state
Bistable - two stable steady states
How are interactions and transcription modelled?
Interactions - Law of mass action
Transcription - modelled by michaelis-menten (by rate of binding of promoter and rate of transcription to predict amount of mRNA)
What is Boolean modelling?
Generates a unitary output from no more than two inputs
- AND, OR, NOT
- each component has two states, on or off
DISCOVERY: That PP2C is regulated by Ca2+ and later found EF hands
What is OnGuard Quantitative Kinetic modelling?
- Use of kinetic models integrating ion channels, pump and metabolites
= Modelling the movement of ions
Can also be used to see how changes in the environment may have an affect
What is the circadian clock and its role?
Endogenous time keeping mechanism that regulates a 24hr cycle
Role: Anticipate environmental changes to optimise physiology
What are the properties of the circadian clock?
- Entrainment by environmental cues
- Has a free running period (constant conditions), but retains 24hr periodic oscillations
- Temperature compensated
What are the 4 key components of the circadian clock?
Entrainment - synchronisation with external time
Circadian oscillator - generating 24hr rhythm
Gating - changes output based on entrainment
Output - comversion of temporal information to physiology
What are diel and circadian rhythms? Difference
Diel - 24hr rhythm of dark and light, with oscillations stopping once exposed to constant conditions
Circadian - 24hr rhythm of dark and light, continues under constant conditions, driven by internal oscillator
What is the free-running period
Duration of the circadian cycle in constant conditions
What can the circadian rhythm regulate?
Leaf angle
Growth
Stomatal movement
Flowering
General gene expression
What are the requirements for an oscillator gene? (4)
- Must oscillate in the circadian period
- Mutation leads to circadian defect
- Control expression of other genes, but also self-regulate
- Clamping of genes should stop the clock
Describe the function and role of TOC1
Timing of Cab1
- Short period mutant
- Nuclear localised
- Not part of the light signalling component
- Encodes a pseudo-response regulator (PRR)
ACCUMULATE in the EVENING, repressing CCA1 and LHY
Describe the role of CCA1
Circadian Clock Associated 1 (CCA1)
- MYB like transcription factor, requires Pi by Casein Kinase 2 for proper function
- Binds to initiate photosynthesis and morning-related genes
- Represses TOC1, PPRs, LUX and CCA1 (self)
- OE leads to disruption of the circadian cycle
ACCUMULATE in EARLY MORNING
Describe the role of LHY
Late Elongated Hypocotyl (LHY)
- Similar to CCA1, also a TF and can form heterodimers
- Regulates its own expression
ACCUMULATE in EARLY MORNING
What mediates red and blue light entrainment into the circadian cycle?
Red = phytochromes (PhyA and PhyB)
Blue = cryptochromes (ZLT), containing FAD. Also found to interact with TOC1
Describe the single loop model of the circadian rhythm and its inaccuracies
- TOC1 accumulates and indirectly promotes expression of CCA1 and LHY.
- CCA1 and LHY negatively regulates TOC1 = negative feedback
- Phytochromes sensing red light, influence CCA1 and LHY expression
NEGATIVES - Oversimplication
- cca1 and lhy double mutant, another gene is present for TOC1 oscillations
- delay for TOC1 activation morning complex
What is the role of gigantea (GI)?
Peaks in the evening, forms part of circadian oscillator
- helps maintain the correct phase of the clock by interacting with ZTL in the presence of blue light and stabilises it
- OE analysis changed period length
- role in conserving energy during extended periods of darkness
- Can also bind FKF1 in blue light, releasing repression on CONSTANS caused by CDF1= promote flowering
Describe the importance of sucrose to circadian regulation
Metabolic feedback to the circadian clock
- Bridging photosynthesis and presence of light to darkness and respiration
- Regulates genes expression of CCA1, LHY and TOC1
- In low sugar, expression of GI is altered
GI being important to regulate energy uses, and conserve during extended periods of darkness
What is the role of TOR?
Target of Rapamycin
= kinase that helps regulate response to sucrose and other nutrients
- regulating cell growth and division
How does TOC1 effect Circadian clock associated (CCA1) and Late elongated hypocotyl (LHY)?
- Suppression facilitated by interactions with PRRs
- Maintaining the correct timing
Are toc1 and ztl short or long period mutants?
toc1 short period mutant, does better in short
ztl long period mutant
How does the circadian rhythm regulate immunity?
Growth in phase with pathogens, such as loopers = greater resistance
Growth out of phase leads to greater susceptibility
e.g. Loopers more likely to feed in light, coincides with peak jasmonates
How does the circadian clock vary at different latitudes?
- Higher latitudes have greater variation in light intensity and quality
- However lower, closet to the equator there is minimal change between seasons
- Also less temperature changes throughout the year
What is phase advance and phase delay?
Phase advance - internal clock shifted early relative to normal
Phase delay - internal clock shifted later, relative to normal
- Pulses of light administered in the morning leads to phase advance, while pulse in the middle of the day leads to phase delay
What is gating?
Activity of a gene is restricted to certain times of the day within the 24hr period
Describe dawn light regulation and the main transcription factor involved
Far Red Elongated Hypocotyl 3 (FHY3)
- acts downstream of PhyA
- Gated by the circadian oscillator, only activates CCA1 at certain times of the day
- Antagonised by TOC1
Describe dusk light regulation and the transcription factor involved
Modulation of ZTL and COP1 activity
In light conditions: PHR and CRY inhibit COP1 and GIGANTEA inhibits ZTL
Dark conditions: ZTL and COP1 mediate degradation of clock oscillatory proteins
How does temperature entrain the circadian clock?
- Pulses of higher temperature causes a phase shift
- ELF3 is degraded leading to an increase in PRR7 and a decrease in morning phase genes (CCA1/LHY)
- Morning pulse until 6pm leads to a phase delay
- Between 6pm to 24:00 leads to phase advance
What is the evening complex?
- ELF3 + ELF4 and LUX (DNA binding protein)
- Required for temperature entrainment (suggested ELF3 to be main part)
- Suppresses expression of PRR7 and PRR9
How were sugar shown to entrain the oscillator?
- Inhibition of photosynthesis by CO2 free area can lengthen the circadian period
- Dark and addition of sucrose, changed circadian cycle and expression of CCA1/LHY
- prr7 + 9 mutants are insensitive to sucrose. Sucrose found to modulate these morning genes
What are the effects of low energy on regulation of PRR7?
- Low energy activates SnRK1 (KIN10/11)
- KIN10/11 phosphorylates bZIP63 at serine residues causing conformational change
- bZIP63 accumulates in the nucleus and binds to promoters (promotes ATG8, Sucrose synthase and indirectly inhibits TOR)
= Suppresses PRR7, conservation of energy
What is the role of PIF4?
bHLH transcription factor that activates growth genes and auxin responsive genes
- expression oscillates
- Degraded by PhyB interaction which is light dependent
Mutants have phy have long hypocotyl
What inhibits and what promotes PIF?
Inhibits
- PRR7/9, TOC1, PHYB and evening complex (EC)
- COP1 in light
- DELLA
Promotes
- darkness,
- auxins, BR and
- Stress conditions
- GI
Why is the study of the circadian clock agronomically relevant?
- Regulates metabolism and photosynthesis
CHRONOCULTURE - harnessing the circadian clock to improve crop yield and sustainability. - Change in geographical change, resulting in changes in light which regulates development e.g. tomato cultivation changed
- Also relevant for climate change, change in temperature without significant changes in light resulting in early flowering and reduced growth
What is the effect of temperature on TOC1 and growth?
High temperature stabilises TOC1 and suppresses PIF which reduces growth
- Can also change leaf number, increasing them
What is the N and p fertiliser use efficiency by the plant?
N = 33%
P = 16% - due to properties in soil
Describe the global distribution of fertilisers
- Unevenly distributed across the globe
- Greater production in HICs due to cost of the process, however, a lot of the consumption is elsewhere, e.g. in LICs
- China and India both produce and consume a lot
Name some examples of biological N fixation
Rhizobia - also have a nitrogenase enzyme, from Sym plastid. Use leghemoglobin from the plant for oxygen for respiration
Cyanobacteria - have heterocysts and nitrogenase enzymes that convert N to ammonia. Followed by GOGAT for fixing
Describe examples of large scale nutrient cycles?
Extinction events resulting in large scale death of organisms and material for decomposition
- End-Permian caused by volcanic events at Siberian traps
- End-Cretaceous 66Mya dinosaurs
How do plants adapt to changes in nutrient availability?
- Changes to root architecture
- Changes to root exudates
- Increase in carboxylates to compete with Pi - Interaction with microbes
Describe the interaction with ectomycorrhizal fungi
- Evolved more recently and restricted to 2% of all species
- Forms a Hartig net around the root for nutrient exchange
- Doesn’t penetrate the cell wall
e.g. Pine seedling grown in symbiosis with the ectomycorrhizal fungus Suillus bovinus
Describe the phenomenon of N symbiosis
- Evolved 80-100Mya
- Cross-over with symbiosis pathway due to CSSP
- Symbiosis carried out by Frankia and Rhizobia with legumes
What are the 4 pathways for N signalling?
- Local N perception
- Long distance for depletion (CEPs)
- Long distance for replete (hormones, cytokinins)
- Shoot derived signals
What is the evidence for systemic signalling of nutrient deficiency?
- Split root system, with stimulus being applied on one side and monitoring overall changes
- Varying signals of replete and deplete nutrient conditions, which then result in proliferation of roots in replete conditions and suppression in deplete half
Describe the signalling pathway for N deficiency
- Productions of CEPs
- Travel up the roots to the shoot where they bind to CEP receptors
- Activation of CEP downwards (CEPD) peptides that travel down to elicit response
- Prioritise N uptake up increasing the expression of high affinity nitrate transporters by CEP induced phosphatases (CEPH)
How does CEPH have a targeted action, and upregulate in certain N conditions?
CEPD induced phosphatases
- More active under N deplete conditions to upregulate HA transporters
- Activated by dephosphorylation at the Ser-501 residue
- Requires integration of local N starvation signalling
Describe the role of NRT1.1 in nitrate sensing and experiments that were carried out
- Mutation in certain amino acids leads to disruption in transport
- Found that changes in gene expression still occurred, mediated by NLP7 that was still activated due Ca2+ influx that activates CPK10/30/32
- NRT2.1 expression which acts downstream was still increased, proving role of NRT1.1 in N sensing
Describe the downstream signalling from the sensing function of NRT1.1
- Senses low levels of nitrate
- Triggers Ca2+ influx into the cell
- Activates CPK10/30/32 and CBL1/9
- CBL1/9 activates CIPK23, phosphorylates NRT1.1 to become HA take up more nitrate
- Activation of NLP7 by phosphorylation at Ser-205 by CPK10/30/32 leading to retention in nucleus
- NLP7 promotes expression of primary nitrate response genes
Describe the transport function of NRT1.1
Low nitrate - NRT1.1 transports auxin away from root tip so no growth.
High nitrate - NRT1.1 transports nitrate instead, so auxin can build up and cause lateral root growth. Generating greater surface area for N take up.
What promotes foraging by the roots for nutrients?
Changes to architecture triggered by presence of cytokinin
- cytokinin synthesis mutants, unable to forage for N
- also leads to downregulation of HA transporters by cytokinin to save energy
How can nitrate signal integrate with carbon signalling?
In normal conditions phosphorylation of NLP7 is via CPK10/30/32 at a Ser-205 residue resulting in nuclear localisation
In either C or N deficiency, phosphorylation of NLP7 is by SnRK1 (KIN10) at different serine residues which leads to cytoplasmic localisation
What is the role of NLP7?
Transcription activator and intracellular nitrate sensor = activity regulated by its localisation
- In high N it is retained in the nucleus
- Nitrate capable of binding directly to conserved domain in NLPs
- Targets nitrate responsive genes, such as NRT2.1
- Links auxin signalling and nitrate signalling in the root cap
Describe the phosphate starvation response
- Activation of PHR transcription factors
- Activate PSI genes promoting phosphate uptake such as PHO1, PHF1 and PHTs
- Also activates SPX as a feedback loop, which are important in sensing InsP and binding to PHR and prevent it from functioning
- Regulation downstream is via microRNA, 399 for PHO2 and miR827 targetting NLA1
- Also suppresses immunity to support symbiosis
What are the methods in which PHO2 are regulated?
- Post translation by phosphorylation by casein kinase 2 that decreases stability
- Post-transcriptionally by miR399, that is inhibited itself by IPS1.
How does strigolactone synthesis effect phosphate uptake?
SL inhibits shoot branching, mutant had more branching
- Cross over with symbiosis, PHR2 able to bind and activate SL genes
SL important for symbiosis establishment, triggering hyphal branching etc.
What expression analyses show that PHR2 is involved in AM symbiosis?
- PHR2 predominantly expressed in the epidermis and steele
- AM colonisation mainly occurs in the cortex, and GUS reporter showed it localised there when colonised
- Also shown to bid to NSP2, ARK1 and other AM genes
- mutant phr2 showed less colonisation, and 70% reduction in AM-related genes
How can plants distinguish between friend and foe for fungi?
Foe - immunity triggered upon detection of peptidoglycan, activating WRKYs in the nucleus
Friend - lipochitooligosaccharides (LCO) sensed by LysM receptors, activating CCaMK
(also in relation to drop in immunity, upon nutrient stress which leads to symbiosis)
What are the different types of SLs?
Canonical
- regulate AM symbiosis, mutants have delayed symbiosis
Non-canonical
- regulates shoot branching
Describe the SL and SMAX1 signalling pathway in AM symbiosis
SMAX1 acts as a repressor of SL signalling
1. Upon SL perception by D14L, SMAX1 is targeted for ubiquitination
2. Releases inhibition of SL-responsive genes
What is autoregulation of nodule symbiosis?
- Spreading out of nodules to prevent overoccumulation
- Regulated by Too Much Love (TML)
1. Clavata3-like (CLE) acts as a signal
2. Binds to CEP receptor (HAR1)
3. Promotes TML to suppress nodules - TML regulated by miR2111
What is the role of FUN?
Encodes a transcription factor that has a sensor domain binding Zinc
High Zn/ Low nitrate -> filamentous form that removes suppression of symbiosis, promoting nodule formation. FUN is multimeric
Low Zn/ High nitrate -> monomeric FUN, upregulates NRT2.1 and HO1 (haem oxygenase that degrades leghaemoglobin to cause nodule senescence)
What are phytohormones?
Chemical messengers that co-ordinate growth and development of cells. In plants, not centralised released, all cells are capable of releasing chemicals
- Examples: Auxin, Cytokinins, Gibberellins, ABA and ethylene, JA and SA
With synthesis being tightly controlled
Describe the movement, reception and transduction of hormones
Movement - through xylem or phloem, or gaseous, or through transport proteins
Receptors - membrane bound, or soluble across the membrane
Transduction - Phosphorylation/ Dephosphorylation. Transcriptional changes (longer), or non-genomic direct-binding changing conformation or channel activity
Describe the structure of the auxin inducible gene
- Auxin Responsive elements (ARE)
- To which Auxin Responsive Factors (ARFs) bind to, promoting transcription - Rest is the gene
- Aux/IAA a transcriptional repressor, capable of binding to ARF and inhibiting it
What genes were discovered to be part of the ubiquitination of Aux/IAA?
- AXR1 encodes subunit for RUB1 activating enzyme
- TIR1 encodes an F-box protein conferring specificity to Aux/IAA.
- from Co-IP studies that found binding in an auxin dose responsive manner.
What is the role of AXR3, what did gain-of-fucntion mutant analysis show?
AXR3 member of Aux/IAA transcriptional repressor family
- mutant leads to auxin resistant root elongation (no response to auxin)
Describe the structure of the Aux/IAA family
Domain I - contains EAR domain that recruits TOPLESS, for transcription repression
Domain II - important for degradation, GUS destabilised in WT, compared to mutant axr3
Domain III - involved in dimerisation with ARFs, mediated by PB1 domain
Domain IV - also involved in dimerisation, contains PB1 domain
What is the role of TOPLESS (TPL)?
Contains WD40 which complexes with HDACs
- de-acetylase which targets histones
-deacetylation of histones results in tighter DNA packaging
- preventing transcription of a gene
What is skotomorphogenesis and photomorphogenesis?
Skoto- plant response to the dark e.g. etiolation
Photo - plant response to light
What are the 3 key receptors for the perception of light (red.blue and UV)
- Phytochromes
- Cryptochromes
- UVR8
What is the role of COP1?
Constitutive photomorphogenesis
- mutant phenotype has short hypocotyl
- an E3 ligase, targeting receptors (phyA as well as HY5)
What is the role of HY5?
Transcription factor repressing cell elongation, nutrient acqusition genes and photosynthesis genes
- mutant has an elongated hypocotyl
What could be done to test whether growth of the hypocotyl is due to the presence of photosynthate, or in response to light?
- Growth in CO2 free air, so unable to carry out photosynthesis
- Knockout genes in the photosynthesis pathway so unable to produce photosynthate
- Growth in different sucrose conditions
What is the effect of different sucrose concentrations on plant growth?
- Different sucrose concentrations didn’t effect hypocotyl growth, but did affect root growth
- in low sucrose conditions, in cop1 mutant growth of the root differed in light and dark conditions
= Plant invest more energy into the hypocotyl until it is able to photosynthesis, then starts growing root system, so shoot less affected by sucrose conditions as it is prioritised
What is the role of PIF4?
TF responsible for the growth seen in the dark, skotomorphogenesis
- Mutant pif4 shows lack of growth
- Increases in during the night
- Suppressed by HY5
- Also can be suppressed by Pfr
Describe how red light is detected
- Use of phytochromes that detects light via bilib
- phyB mutant etiolates
DARK = Pr inactive
LIGHT = Pfr active
Detection of light by Pr leads to conversion to Pfr, which then translocates to the nucleus and inhibits PIF from promoting growth
How is blue light sensed (not in circadian clock)?
- By cryptochromes that contain the Flavin Adenine Dinucleotide (FAD)
Active - by phosphorylatin
Deactivated - by dephophorylation - Activates HY5 to suppress PIFs
How is blue light sensed for circadian clock entrainment?
By ZEITLUPE (ZTL)
- Contains Flavin Mononucleotide (FMN)
- Indirectly promotes PIF degradation
- Also targets TOC1 for degradation (which is present in the evening and suppresses morning LHY/ CCA1)
How is UVB sensed by the plant?
- Detection by UVR8 which has tryptophan
MONOMER - active
Dimer - inactive - Dimer forms from ionic interactions between positive and negative charges
Method of action - Binds to the pocket of COP1 so that HY5 can’t bind and be degraded. So HY5 is free to inhibit PIFs
Describe the role of Gibberellic acid signalling on growth
DELLA is present as a transcriptional repressor when there is no GA
1. GA binds to GA receptor GID1, conformational change occurs
2. GID1 binds to DELLA targetting it for proteolysis
3. Transcription factor no longer suppressed
What are the similarities and differences between Auxin and GA signalling?
Similarities
1. Presence of suppressors Aux/IAA and DELLA that are then targeted in presence of auxin and GA, respectively
2. Involvement of ubiquitin and 26S proteosomal degradation
3. Differences
Auxin - TIR1is the receptor and E3 ligase
GA - GID1 is the receptor, that then recruits SLY as an E3 ligase
Describe the pathway of Brassinosteroid signalling (6)
- BR is detected by BRI1, that binds to coreceptor BAK1
- Co-phosphorylate each other
- Activation of BSKs
- BSKs phosphorylate BSU1, a phosphatase
- BSU1 acts on BIN2, dephosphorylating it and rendering it inactive
- BIN2 no longer able to inhibit transcription factor BZR1/ BES1
Describe the BAP-D module
Brassinosteroid- auxin- PIF DELLA module
- ARFs, PIFs and BZR1 are bHLH TF
- can complex together and regulate transcription at the the G-Box and GCEs
- DELLA can inhibit all 3
Suggest how hormonal signalling pathways overlap to regulate growth
- PIFs that are regulated by the BAP-D module feedback and regulate GA, BR and auxin signalling (via YUC)
- Use of Ca2+ as a secondary messenger to regulate downstream targets
- Auxin promotes GA biosynthesis and GA affectin PIN localisation, for auxin transport, in rice
Describe the ways in which hormone levels can be detected
- Chromatography and Mass spec
- Separation then detection - ELISA - generating antibodies against the hormone
- Fluorescent biosensors- allows for real time imaging of hormone concentrations and gradients
e.g. GA gradient, higher conc in the zone of elongation compare to the sub-apical region
What are some genes that regulate hypocotyl elongation downstream of BZR/BES/ARFs and PIFs?
- IBH1 acts as a suppressor for elongation
- PRE1 (bHLH TF) with no DNA binding domain, promotes elongation
- HBI1 - growth promoting bHLH TF, more present in larger cells
Describe the structure and function of bHLH transcription factors
basic helix loop helix
- Helix 1 with N terminus is crucial for DNA binding
- Helix 2 with C terminus crucial for dimerisation
= Bind to DNA and regulate transcription, often acting as dimers
How can Ca2+ encode signal specificity?
- Different signals with different graded responses
- Spatial and temporal dynamics
- Different cell types for different responses
How can specific gene expression be targeted to a cell?
Enhancer trapper technique
- Transgene contains
1. enhancer detector element (e.g. GAL4)
2. Minimal promoter (TATA) fused to that
3. Resistance/ Marker gene
4. Aequorin (desired expressed gene)
5. Terminator
- So expression only occurs if detection of the endogenous enhancer (cell specific) is present
How does external levels of calcium affect Ca2+ oscillations?
Increase in concentrations can lead to asymmetrical oscillations
Low concentrations may not be enough to start oscillations
Describe the importance of Ca2+ oscillations? Are they always necessary?
- In the tip of pollen tubes, removal of oscillations had no effect
- Transient spikes are also important, for example in an immune response
- General aspects of the oscillation, such as the frequency, amplitude and duration commonly have an effect on signalling and provide specificity
- Frequency of oscillations doesn’t seem to affect stomatal closure, however, did effect the ability to re-open them. Lower freq able to open them better
What is the role of cyclic ADPR on calcium oscillations?
- Important for it to be cyclic, addition leads to oscillations and stomatal closure
- Made from NAD+ by the enzyme CD38
1. Activates TPC1 channels on the tonoplast
2. Ca2+ increases in the cytosol and causes inhibition, resulting in an equilibrium being reached
What is the role of IP3 on Ca2+ oscillations?
- Promotes the presence of oscillations
- Addition of Phopholipase C inhibitor reduces the amount of IP3 and a lack of oscillations
Describe the importance of DET3, and where it is located.
- Vacuolar ATPase responsible for generating the membrane potential
- Mutant det3 unable to generate potential so couldn’t generate the Ca2+ oscillations
- ABA induced closure not as affected as can directly act on SLAC and KAT, and channels on the plasma membrane?
Why is the affinity of the sensor also important for Ca2+ transduction?
- Differences in affinity will sens spikes differently
- High affinity will generate a larger fuller response and not detect it as a spike, while a lower affinity sensor would
- So same oscillation with different sensor can lead to a different response
What are the different short and long term oscillatory mechanisms regulating stomatal closure and aperture?
Short term- regulated by CPKs that phosphorylate and alter channel activity, also the role of ROS in regulating HACCs
- Mutant cpk unable to close stomata due to inability to activate SLAC
Long term - cpk mutant unaffected and not sure about mechanism
What is the interaction between CPK and GORK1?
- CPK21 can phosphorylate GORK1, which in turn is activated by 14-3-3 in salt stress
- Studies showing enhanced K+ efflux in greater concentrations of 14-3-3, and found that CPK21 may form the bridge linking them
- CPK21 and 23 activity enhanced in the presence of 14-3-3
How do plants avoid shade?
Detection of red light that is transmitted through leaves
- Detected PhyB that is activated (Pfr)
Results in hypocotyl and petiole elongation to grow out of the shade
- This is achived by auxin accumulation to promote growth, single cell sequencing found differential regulation in different cell layers
How do plants respond to thermogenesis? (General and specific effects)
Warmth = promotes growth
Cold required for breaking seed dormancy and flowering (winter before spring)
General - alterations to the rate of enzymatic reactions as a result of changes in temperature
Specific - changes to the end goal e.g. size or length of an organ, or number of leaves
How is thermogenesis integrated with PIF and gene expression?
- Via Pfr inactivation (conversion to Pr) due to thermal reversion at higher temp
- Inhibition of ELF3 (part of EC), that is responsible for suppressing PIF4
= Promote growth in warm temperatures
Describe the evidence that shows that PhyB is a thermo and light sensor
Experiment monitoring the transcript levels of ATBH2 a transcription factor promoting hypocotyl elongation, downstream of PIF4
1. At 20°C normal increase in ATBH2 levels occurs at the end of the night, this is due to the half life of Pfr that turns into inactive Pr due to the lack of far red light
2. At 27°C increase in ATBH2 is more rapid at night, due to thermal reversion of Pfr into Pr
- Mutant PhyB did not respond to temperature. So the changes seen between light and dark and different temperatures supports the notion that PhyB acts as a sensor for light and temperature
What is the evidence/ How is ELF3 regulated by temperature?
Experiments looking at light defraction found that ELF3 goes through phase transition
At different temperatures the protein is folded differently, at higher temperatures the solution is less diffuse as folding is more organised
- Undergoes liqui-liquid phase separation (LLPS) that means it’s no longer able to bind and inhibit PIF4
How does the plant distinguish between low light and night?
Circadian clock gating
- Role of circadian clock to accumulate EC in the evening and suppress the mRNA of PIFs
How do plants handle sunflecks?
PhyA and PhyB important in sensing light and responding rapidly to make the most of it
- Increase in HY5 transcript after 2hr of sunfleck, resulting in suppression of growth
- Mutant in phyA/B continued to grow as if it dark conditions, while cry mutant didn’t suggesting PHYA/B is important
How do plants prevent self shading?
- Basal senescence - removing old leaves found at the bottom
- Phyllotaxis - changes in lead arrangement
- Hyponasty - auxin movement from tip to petiole to promote stem elongation
- Lower R:FR indicative of shading shifts phytochrome activity
+ Tip better at detecting shade, which would occur from other plants
How is Calcium efflux carried out to restore the baseline concentration of Ca2+ in the cytosol?
- Requires energy as it is moving against the concentration gradient
- Ca2+ ATPases
- Ca and H+ antiporters (CAX)
Describe the importance of asymmetric hormone distribution
Phototropism and Gravitropsim, also important for organ developing as gradients are needed for apical-basal axis
- auxin accumulation on the shaded side, leading to cell elongation and bending towards the right
- Antagonistic roles in shoots and roots for gravitropism, gradient created by statoliths causing PIN rearrangement and changing auxin distribution. PIN rearrangement could potential be due to NPH3
What receptors detect light and cause shoot bending?
PHOT1 and PHOT2 (Phototropin)
- Have a LOV1/2 photosensory domain containing a Flavin Mononucleotide (FMV) that detects blue light
- Ring like structure that undergoes conformational change and 90° rotation
- Results in kinase domain (of PHOT1) activation and Pi of PIN to re-localise
How do plants respond to soil compaction?
- Mechanical stress is sensed, oxygen deficiency and altered water availability
- Mechanosensitive channels are activated, and hormone production is altered
- Increase in ethylene production and changes to auxin distribution limiting root tip growth
Changes
- Inhibition of root growth
- More curved apical hook (protect meristem)
- Swelling of hypocotyl
Describe the interaction between growth and immunity signalling pathways in plants
Hypocotyl specific BAP-Inducible (HBI) and flg22 from immunity signalling
- Regulate an overlapping set of genes but antagonstically
- For example Flg22-Induced Receptor Kinase 1 (FRK1) expression is reduced in HBI-Ox
What are the two different types of ARFs and their roles?
Class A = Activator
Class B = repressor
- Allowing for diversity of responses
Describe the phylogeny of ARFs, Aux/IAAs and TIR1
The evolutionary divergence from Marchantia to Arabidopsis illustrates the increasing complexity of regulatory networks that have evolved over time
ARF
- DNA binding site shows ultra-conservation
- Varying affinities between ARFs and their promoters
Aux/IAA
- Greater number of genes found in Arabidopsis compared to Marchantia, suggest increase in complexity
- Different Aux/IAA have different half lives
TIR1
- Greater number in A.thaliana
- Redundancy, between them as mutations in all can be lethal
What are the reasons for auxin having so many different roles?
- Diversity in receptors e.g. ARFs, Aux/IAA and TIR1 that can lead to different combinations and different responses
- High degree of cross-talk with other hormones so can have many roles e.g. in hypocotyl elongation and lateral root formation
- Evolutionary conservation and diversification, mutations leads to unfavourable phenotypes so pathway has been conserved
Describe the extracellular perception of auxin
- Auxin detected ABP1
- Activates TMK1 that phosphorylates AHA2
- AHA is a proton pump that acidifies the apoplast
- Promotes cell growth and elongation independent of TIR1 mediated transcriptional regulation
Describe how phototropism occurs
- Light is sensed by photoreceptors (PHY, CRY and UVR8)
- Photoreceptors can:
a. UVR8 monomer moves to the nucleus and supports HY5 suppression of PIFs
b. Photoreceptors alter auxin signalling (PINs)
c. Photoreceptors alter auxin perception (Aux/IAA and TIR1)
d. Photoreceptors can increase GA synthesis to repress growth-suppressor DELLA - b. detection of blue light leads to PHOT1 autophosphorylation, dephosphorylation of NPH3 and removal of PIN3 from membrane on illuminated side
c. changing TIR1 transcript levels in different light, regulating ability to detect and perceive auxin
Hypocotyl photomorphism - Activation of ARF7/19 to bind to ARE in SAUR19
- Expression of SAUR19 leads to inhibition of PP2C, increasing Pi of the proton pump (AHA2) = activation
- Acidification and cell wall remodelling in the epidermis
Describe the process of apical hook opening
Auxin inhibits apical hook growth
- This is due to PP2C preventing AHA2 acidification and PP2C is activated by ARF7, and because it acts in a different area has a different effect
OPENING
- Occurs when light depletes auxin
List morning, day, afternoon and evening phase components
Morning: CCA1, LHY
Day: PRR9 and 7
Afternoon: RVE8
Evening: TOC1, EC (LUX + ELF3 + ELF4)
What is the role of Deetiolated 1 (DET1)?
Helps CCA1 and LHY repress TOC1
DET1 acts a chromatin remodeller, altering gene expression
Explain how CCA1 and TOC1 can undergo post-transcriptional and post-translational regulation
CCA1- changed by alternative splicing
CCA1α generates a fill read through, CCA1β generates a truncated protein. With CCA1α being better for a cold stress response
TOC1- can be targeted for ubiquititation by ZTL in blue light
What is the metabolic day length measurement (MDLM) system and its relevance to the circadian cycle and response to certain stimuli?
System that measures photosynthetic period, and not just photoperiod.
- Has an effect on photoperiodic growth and causes seasonal expression of genes
- MLDM contributes to entrainment, helping plants recognise time and transition between phases, such as flowering
- Sugar sensing signals that are important for MLDM are also clock gated, differing in their response in dark and light conditions
= LINK METABOLIC PROCESSES WITH CIRCADIAN REGULATION
How does day length regulate flowering?
CO-FT system
- CO is a TF that promotes the transcription of FT that promotes flowering
- CO protein is degraded in dark conditions, and COmRNA peaks in the latter half of the day
- In long days, peak of transcript coincides with light, so FT increases and so does flowering.
- However, in short days, peak of CO transcript occurs when it’s dark so the protein is degraded and unable to promote FT
Describe the relationship between PIFs and PRRs
TOC1/ PRR1 cooperates with EC to suppress PIF4 and PIF5
- PRR9/7/5 found to directly interact with PIFs
- Regulating expression throughout the 24hrs
What are the responses of plants to different cold treatments? Cold shock, gradual chilling and non-damaging cold
Cold shock - Stunted growth, inhibition of photosynthesis and formation of lesions
Gradual chilling - hardening of leaves
Non-damaging exposure - allows for acclimatisation, and increased production of ABA
How plants distinguish between symbiosis and immunity signalling? (6)
- ROS production - greater ROS in immunity, although transient in symbiosis
- Nutrient status - low nutrient status = suppressed immune system
- Signalling molecule - LCOs v chitin
- Signalling receptor - different downstream signalling for different effectors
- Location of Ca2+ spike - symbiosis nuclear, immunity cytoplasmic
- Hormonal crosstalk - JA and SA for immunity, cytokinin for symbisis
What is an example of an engineered light-gatedp channel in plants?
BLINK1 - formed by fusing LOV2Jα domain from Avena sativa to a virus K+ channel
- Introduced into guard cells of Arabidopsis to see the effect on stomatal conductance
- Increase rate of stomatal closure and aperture, improving water use efficiency and increasing biomass
- Calculated by measuring membrane potential (patch clamp) and stomatal conductance (gas exchange)
Papanatsiou et al. 2019
What is the triple response of the hypocotyl shoot and why is it important? And how is it achieved?
- inhibit hypocotyl and root elongation
- more pronounced curved apical hook
- radial swelling of hypocotyl
= Protect the shoot meristem from mechanical damage when pushing up through the soil
1. Increase in ethylene production
2. Activates EIN3/EIL1
Triggers
3a. PIF3 to suppress chlorophyll formation, to prevent photo-oxidation when breaching the surface
3b. Regulation of growth by Ethylene Responsive Factor (ERF1)
+ Increase in ethylene and associated gene expression in deeper soil
How does the evolution auxin signalling pathway generated both specificity and diversity?
Specificity (5)
- Different DNA promoter binding affinities for different ARFs
- Retain specificity of binding at the DNA binding domain that is highly conserved
- Spatial and temporal expression specific to response (also generates diversity)
- Different half lives of Aux/IAA
- Specificity is generated by the heterodimer formations and the interactions between all parts of the signalling complex meaning auxin can regulate many physiological factors
Diversity (5)
- Specialisation to have different function iaa17 mutant had no root hair, while iaa3 had long root hair
- Different combinations of ARFs and Aux/IAAs
- Many different ARFs and Aux/IAAs in higher order plants (A.thaliana comparison to Merchantia)
- TIR/AFB evolution targetting Aux/IAA allows for dose-dependent response to auxin
- ARFs can work with receptors from other hormones = crosstalk
How is gravitropism and phototropism signal integrated?
PIN relocalisation resulting in auxin gradients (due to gravitropism and phototropism)
1. Auxin increase removes Aux/IAA inhibition of ARF7/19
2. ARF7/19 bind to promoter of SAUR19
3. Asymmetric SAUR19 leads to growth response
4. SAUR19 inhibits PP2C from de-Pi AHA2 (H+ pump)
5. Increase Pi of AHA2 = acidification and membrane remodelling
How is thermomorphogenesis and phototropism interlinked?
- Regulation of HY5
- Increase in HY5 transcript in cold conditions, even at night which suppresses growth by suppressing PIF4 - Direct regulation of PIFs by photoreceptors such as PHYB
- UVR8 binding to COP1 to inhibit its degradation of HY5 resulting in photomorphogenesis
