M1: Signalling Flashcards

Question

How can Ca2+ channels be located/ discovered?

Answer 1

1. Patch clamp electrophysiology - Discovery of TPC1 2. Planar lipid bilayers - vesicles bind to artificial membrane and measure changes 3. Genomics looking at homology - Discovery of GLRs and CNGCs 4. Genomics looking at mutant screens

Answer 2

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)

Answer 3

1. 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 2. 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)

Answer 4

1. 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 2. 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)

Answer 5

Soluble proteins that can bind to Ca2+ and localise to a membrane facilitating transport

Answer 6

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

Answer 7

CAX1 - low affinity antiporter of Ca2+/H+ across the vacuole membrane - autoinhibited by Cax interacting proteins instead of CaM

Answer 8

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

Answer 9

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

Answer 10

1. Calmodulin (CaM) 2. Calmodulin-like (CML) 3. Ca2+ dependent protein kinase (CDPK) 4. Calcineurin B-like (CBL)

Answer 11

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

Answer 12

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

Answer 13

- 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

Answer 14

- 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

Answer 15

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)

Answer 16

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

Answer 17

- 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

Answer 18

- 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

Answer 19

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)

Answer 20

1. Fluorescence labelling, found 4 steps of fluorescence quenching 2. Repeat but with two different dyes, found two steps of quenching

Answer 21

1. Phosphorylation by CPK32 2. Regulation by heteromer formation with CNGC7/8 3. Activation by Calmodulin, promoting heteromeric channels 4. Ca2+ presence

Answer 22

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

Answer 23

Kd (dissociation constants) differ Lower indication stronger interactions - Analysis suggest CNGC8 stronger and more stable interaction

Answer 24

- 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

Answer 25

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

Answer 26

- 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

Answer 27

1. Binds to PYL receptors 2. Inhibits PP2C and activates SnRK2/OST1 3. Phosphorylates RBOH, SLAC1, KAT1, H+ ATPase and HACCs 4. HACC leads to [Ca2+]cyt changes - Targetting CNGC 5,6,9,12 5. Activation of K+ efflux channel due to depolarisation 6. Loss in turgor of guard cell so stomatal closure

Answer 28

1. FLS2 (a PRR) binds to flg22 2. Undergoes conformational change and binds to co-receptor BAK1 3. Phosphorylation occurs, leading to activation of BIK1 4. BIK1 phosphorylates many targets, one of them being OSCA1.3 to promote Ca2+ channel opening. Also primes CNGC4 for activation 5. Promoting stomatal closure to prevent pathogen entry

Answer 29

1. 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 2. CNGCs - gated by cAMP and cGMP 3. IP3 and InsP6 pathway - regulating calcium channels

Answer 30

- 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

Answer 31

1. Release of eATP 2. eATP perceived by DORN1 that controls MAPK and Ca2+ response 3. Results in a Ca2+ influx, requires CNGC2 (but no CNGC4), from mutant analysis - Indirect activation by the production of cyclic nucleotides e.g. cAMP 4. Initial influx of Ca2+ through CNGC2 leads to activation of Ca2+ induced Ca2+ release e.g. activation TPC1

Answer 32

1. Detection by PRR and co-receptor binding e.g. BAK1 2. Signal tranduction by GLRs (Ca2+ channels) and CNGCs 3. Release from intracellular stores (TPC1) 4. Downstream targets includes the production of ROS, hormone production and the MAPK cascade activation

Answer 33

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

Answer 34

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

Answer 35

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

Answer 36

- 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

Answer 37

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)

Answer 38

Depolarise the membrane away from -120mV to 0mV Inhibit the H+ proton pump and K+ influx channel, promoting K+ efflux

Answer 39

1. Screening to find chemical that acts in a similar way to ABA (inhibit seed growth) and found Pyrabactin 2. Mutant analysis looking for the receptor - Found that quadruple mutant for the receptor was unable to respond to ABA

Answer 40

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

Answer 41

1. In vitro binding assays 2. Co-immunoprecipitation assays 3. Bi-fluorescence complentation using YFP

Answer 42

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

Answer 43

1. ABA binding to PYR removes suppressive activity of PP2C on OST1 2. Autophosphorylation of OST1 occurs 3. Activation/ Priming of downstream targets to increase Ca2+ concs to induce stomatal closure e.g. RBOH, SLAC1

Answer 44

- 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

Answer 45

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

Answer 46

- 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+

Answer 47

1. Ca2+ leads to TPC1 activation, moves out K+ and Ca2+ 2. TPK1 activated by increased Ca2+, moving out more K+

Answer 48

- 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

Answer 49

1. See if understanding is correct, by regenerating 2. Find new levels of regulation 3. Optimising pathways, find rate-limiting steps

Answer 50

Monostable - one stable steady state Bistable - two stable steady states

Answer 51

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)

Answer 52

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

Answer 53

- 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

Answer 54

Endogenous time keeping mechanism that regulates a 24hr cycle Role: Anticipate environmental changes to optimise physiology

Answer 55

1. Entrainment by environmental cues 2. Has a free running period (constant conditions), but retains 24hr periodic oscillations 3. Temperature compensated

Answer 56

Entrainment - synchronisation with external time Circadian oscillator - generating 24hr rhythm Gating - changes output based on entrainment Output - comversion of temporal information to physiology

Answer 57

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

Answer 58

Duration of the circadian cycle in constant conditions

Answer 59

Leaf angle Growth Stomatal movement Flowering General gene expression

Answer 60

1. Must oscillate in the circadian period 2. Mutation leads to circadian defect 3. Control expression of other genes, but also self-regulate 4. Clamping of genes should stop the clock

Answer 61

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

Answer 62

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

Answer 63

Late Elongated Hypocotyl (LHY) - Similar to CCA1, also a TF and can form heterodimers - Regulates its own expression ACCUMULATE in EARLY MORNING

Answer 64

Red = phytochromes (PhyA and PhyB) Blue = cryptochromes (ZLT), containing FAD. Also found to interact with TOC1

Answer 65

- 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

Answer 66

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

Answer 67

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

Answer 68

Target of Rapamycin = kinase that helps regulate response to sucrose and other nutrients - regulating cell growth and division

Answer 69

- Suppression facilitated by interactions with PRRs - Maintaining the correct timing

Answer 70

toc1 short period mutant, does better in short ztl long period mutant

Answer 71

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

Answer 72

- 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

Answer 73

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

Answer 74

Activity of a gene is restricted to certain times of the day within the 24hr period

Answer 75

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

Answer 76

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

Answer 77

- 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

Answer 78

- ELF3 + ELF4 and LUX (DNA binding protein) - Required for temperature entrainment (suggested ELF3 to be main part) - Suppresses expression of PRR7 and PRR9

Answer 79

- 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

Answer 80

1. Low energy activates SnRK1 (KIN10/11) 2. KIN10/11 phosphorylates bZIP63 at serine residues causing conformational change 3. bZIP63 accumulates in the nucleus and binds to promoters (promotes ATG8, Sucrose synthase and indirectly inhibits TOR) = Suppresses PRR7, conservation of energy

Answer 81

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

Answer 82

Inhibits - PRR7/9, TOC1, PHYB and evening complex (EC) - COP1 in light - DELLA Promotes - darkness, - auxins, BR and - Stress conditions - GI

Answer 83

CHRONOCULTURE - harnessing the circadian clock to improve crop yield and sustainability. 1. Change in geographical change, resulting in changes in light which regulates development e.g. tomato cultivation changed due to mutation in LNK2 causing elongation of circadian period 2. Also relevant for climate change, change in temperature without significant changes in light resulting in early flowering and reduced growth, EC important for entraining temperature 3. Indoor farming - controlled conditions and used to optimise growth. Convergence of photoperiods to allow multiple crops to be grown in the same conditions 4. Biotic interactions- dependent on the circadian cycle, lhy mutants in Medicago had reduced nodule formation (Steed et al., 2021)

Answer 84

High temperature stabilises TOC1 and suppresses PIF which reduces growth - Can also change leaf number, increasing them

Answer 85

N = 33% P = 16% - due to properties in soil

Answer 86

- 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

Answer 87

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

Answer 88

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

Answer 89

1. Changes to root architecture 2. Changes to root exudates - Increase in carboxylates to compete with Pi 3. Interaction with microbes

Answer 90

- 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

Answer 91

- Evolved 80-100Mya - Cross-over with symbiosis pathway due to CSSP - Symbiosis carried out by Frankia and Rhizobia with legumes

Answer 92

1. Local N perception 2. Long distance for depletion (CEPs) 3. Long distance for replete (hormones, cytokinins) 4. Shoot derived signals

Answer 93

- 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

Answer 94

1. Productions of CEPs 2. Travel up the roots to the shoot where they bind to CEP receptors 3. Activation of CEP downwards (CEPD) peptides that travel down to elicit response 4. Prioritise N uptake up increasing the expression of high affinity nitrate transporters by CEP induced phosphatases (CEPH)

Answer 95

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

Answer 96

- 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

Answer 97

1. Senses low levels of nitrate 2. Triggers Ca2+ influx into the cell 3. Activates CPK10/30/32 and CBL1/9 4. CBL1/9 activates CIPK23, phosphorylates NRT1.1 to become HA take up more nitrate 5. Activation of NLP7 by phosphorylation at Ser-205 by CPK10/30/32 leading to retention in nucleus 6. NLP7 promotes expression of primary nitrate response genes

Answer 98

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.

Answer 99

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

Answer 100

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

Answer 101

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

Answer 102

1. Activation of PHR transcription factors 2. Activate PSI genes promoting phosphate uptake such as PHO1, PHF1 and PHTs 3. Also activates SPX as a feedback loop, which are important in sensing InsP and binding to PHR and prevent it from functioning 4. Regulation downstream is via microRNA, 399 for PHO2 and miR827 targetting NLA1 5. Also suppresses immunity to support symbiosis

Answer 103

1. Post translation by phosphorylation by casein kinase 2 that decreases stability 2. Post-transcriptionally by miR399, that is inhibited itself by IPS1.

Answer 104

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.

Answer 105

- 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

Answer 106

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)

Answer 107

Canonical - regulate AM symbiosis, mutants have delayed symbiosis Non-canonical - regulates shoot branching

Answer 108

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

Answer 109

- 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

Answer 110

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)

Answer 111

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

Answer 112

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

Answer 113

1. Auxin Responsive elements (ARE) - To which Auxin Responsive Factors (ARFs) bind to, promoting transcription 2. Rest is the gene 3. Aux/IAA a transcriptional repressor, capable of binding to ARF and inhibiting it

Answer 114

1. AXR1 encodes subunit for RUB1 activating enzyme 2. TIR1 encodes an F-box protein conferring specificity to Aux/IAA. - from Co-IP studies that found binding in an auxin dose responsive manner.

Answer 115

AXR3 member of Aux/IAA transcriptional repressor family - mutant leads to auxin resistant root elongation (no response to auxin)

Answer 116

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

Answer 117

Contains WD40 which complexes with HDACs - de-acetylase which targets histones -deacetylation of histones results in tighter DNA packaging - preventing transcription of a gene

Answer 118

Skoto- plant response to the dark e.g. etiolation Photo - plant response to light

Answer 119

1. Phytochromes 2. Cryptochromes 3. UVR8

Answer 120

Constitutive photomorphogenesis - mutant phenotype has short hypocotyl - an E3 ligase, targeting receptors (phyA as well as HY5)

Answer 121

Transcription factor repressing cell elongation, nutrient acqusition genes and photosynthesis genes - mutant has an elongated hypocotyl

Answer 122

- 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

Answer 123

- 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

Answer 124

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

Answer 125

- 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

Answer 126

- By cryptochromes that contain the Flavin Adenine Dinucleotide (FAD) Active - by phosphorylatin Deactivated - by dephophorylation - Activates HY5 to suppress PIFs

Answer 127

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)

Answer 128

- 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

Answer 129

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

Answer 130

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

Answer 131

1. BR is detected by BRI1, that binds to coreceptor BAK1 2. Co-phosphorylate each other 3. Activation of BSKs 4. BSKs phosphorylate BSU1, a phosphatase 5. BSU1 acts on BIN2, dephosphorylating it and rendering it inactive 6. BIN2 no longer able to inhibit transcription factor BZR1/ BES1

Answer 132

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

Answer 133

1. PIFs that are regulated by the BAP-D module feedback and regulate GA, BR and auxin signalling (via YUC) 2. Use of Ca2+ as a secondary messenger to regulate downstream targets 3. Auxin promotes GA biosynthesis and GA affectin PIN localisation, for auxin transport, in rice

Answer 134

1. Chromatography and Mass spec - Separation then detection 2. ELISA - generating antibodies against the hormone 3. 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

Answer 135

1. IBH1 acts as a suppressor for elongation 2. PRE1 (bHLH TF) with no DNA binding domain, promotes elongation 3. HBI1 - growth promoting bHLH TF, more present in larger cells

Answer 136

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

Answer 137

- Different signals with different graded responses - Spatial and temporal dynamics - Different cell types for different responses

Answer 138

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

Answer 139

Increase in concentrations can lead to asymmetrical oscillations Low concentrations may not be enough to start oscillations

Answer 140

- 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

Answer 141

- 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

Answer 142

- Promotes the presence of oscillations - Addition of Phopholipase C inhibitor reduces the amount of IP3 and a lack of oscillations

Answer 143

- 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?

Answer 144

- 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

Answer 145

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

Answer 146

- 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

Answer 147

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

Answer 148

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

Answer 149

1. Via Pfr inactivation (conversion to Pr) due to thermal reversion at higher temp 2. Inhibition of ELF3 (part of EC), that is responsible for suppressing PIF4 = Promote growth in warm temperatures

Answer 150

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

Answer 151

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

Answer 152

Circadian clock gating - Role of circadian clock to accumulate EC in the evening and suppress the mRNA of PIFs

Answer 153

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

Answer 154

1. Basal senescence - removing old leaves found at the bottom 2. Phyllotaxis - changes in lead arrangement 3. Hyponasty - auxin movement from tip to petiole to promote stem elongation 4. Lower R:FR indicative of shading shifts phytochrome activity + Tip better at detecting shade, which would occur from other plants

Answer 155

- Requires energy as it is moving against the concentration gradient - Ca2+ ATPases - Ca and H+ antiporters (CAX)

Answer 156

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

Answer 157

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

Answer 158

1. Mechanical stress is sensed, oxygen deficiency and altered water availability 2. Mechanosensitive channels are activated, and hormone production is altered 3. 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

Answer 159

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

Answer 160

Class A = Activator Class B = repressor - Allowing for diversity of responses

Answer 161

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

Answer 162

1. Diversity in receptors e.g. ARFs, Aux/IAA and TIR1 that can lead to different combinations and different responses 2. High degree of cross-talk with other hormones so can have many roles e.g. in hypocotyl elongation and lateral root formation 3. Evolutionary conservation and diversification, mutations leads to unfavourable phenotypes so pathway has been conserved

Answer 163

1. Auxin detected ABP1 2. Activates TMK1 that phosphorylates AHA2 3. AHA is a proton pump that acidifies the apoplast 4. Promotes cell growth and elongation independent of TIR1 mediated transcriptional regulation

Answer 164

1. Light is sensed by photoreceptors (PHY, CRY and UVR8) 2. 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 3. 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 4. Activation of ARF7/19 to bind to ARE in SAUR19 5. Expression of SAUR19 leads to inhibition of PP2C, increasing Pi of the proton pump (AHA2) = activation 6. Acidification and cell wall remodelling in the epidermis

Answer 165

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

Answer 166

Morning: CCA1, LHY Day: PRR9 and 7 Afternoon: RVE8 Evening: TOC1, EC (LUX + ELF3 + ELF4)

Answer 167

Helps CCA1 and LHY repress TOC1 DET1 acts a chromatin remodeller, altering gene expression

Answer 168

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

Answer 169

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

Answer 170

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

Answer 171

TOC1/ PRR1 cooperates with EC to suppress PIF4 and PIF5 - PRR9/7/5 found to directly interact with PIFs - Regulating expression throughout the 24hrs

Answer 172

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

Answer 173

1. ROS production - greater ROS in immunity, although transient in symbiosis 2. Nutrient status - low nutrient status = suppressed immune system 3. Signalling molecule - LCOs v chitin 4. Signalling receptor - different downstream signalling for different effectors 5. Location of Ca2+ spike - symbiosis nuclear, immunity cytoplasmic 6. Hormonal crosstalk - JA and SA for immunity, cytokinin for symbisis

Answer 174

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

Answer 175

- 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

Answer 176

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

Answer 177

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

Answer 178

1. Regulation of HY5 - Increase in HY5 transcript in cold conditions, even at night which suppresses growth by suppressing PIF4 2. Direct regulation of PIFs by photoreceptors such as PHYB 3. UVR8 binding to COP1 to inhibit its degradation of HY5 resulting in photomorphogenesis

Answer 179

When the inside of the cell becomes more positive, with respect to the outside of the cell - E.g. stomatal closure as a result of Ca2+ moving into guard cell

Answer 180

1. Mutants in OST1 had constantly open stomata so cooler leaves 2. In vitro kinase activity assay to show that it autophosphorylates