Plant growth regulators: Ethylene, auxins, gibberellins Flashcards
3 part lecture:
- Ethylene – atypical plant hormone gas
- Auxins – hormones and weak plant acids, have a role in regulating plant growth
- Gibberellins – hormones involved in growth regulation across all plant groups
Ethylene lecture:
1.Discovery of ethylene
2.Production of ethylene
3.Physiological effects of ethylene
4.The genetic determination of ethylene signalling
- Discovery of ethylene
Some physiological effects of ethylene on plant tissue
triple response: Neljubow 1901:
Neljubow was growing pea plants in dark conditions the expected results were long stems and yellow leaves but as he continued with his experiments he found the plants began to grow shorter and shorter in height – he was working late into the night burning gas lights (no electricity in 1901) and as it turned out the gas was affecting his experiments
St Petersburg 1901: Neljubow observed that trees prematurely lost leaves when growing next to gas lamps
^ Coal gas = illuminating gas in cities (gas lights) and he realised his lamps may be affecting their growth.
However Neljubow wasn’t able to determine whether the chemical in coal gas was also produced naturally in plants
(1920-30’s) Ripening lemons:
- Lemon growers stored newly harvested green lemons in sheds kept warm by kerosene heaters until they turned yellow.
- They had assumed it was the heat that ripened the lemons however in 1930’s modern electric heating systems were tried and the lemons no longer turned yellow on time.
- The important factor in the ripening process turned out to be a small amounts of ethylene gas given off by the burning kerosene.
^ethylene has a role in ripening fruit, bananas emit a lot of ethylene so are best kept separate from other fruit as in the picture
Plants can respond to very low levels of ethylene. There is very little ethylene gas in the atmosphere this makes it an effective long distance signalling hormone which has very little noise.
2.Production of ethylene
Changes in ethylene and ACC content and ACC oxidase activity during fruit ripening
ACC synthase produces a small molecule called ACC and ACC oxidase oxidises ACC to release ethylene gas. The graph above shows the process of golden delicious apple ripening in days after harvest.
However there is a big difference between causality and correlation.
Dipping the same apple into silver nitrate results in the same chemicals being produced but no ripening occurring as silver blocks the receptor for ethylene – confirming ethylene is necessary for ripening
Ethylene production and respiration
fruit ripening process is cataclysmic – fruits give off CO2 as they ripen, at a certain point ethylene production stimulates a huge spike in respiration resulting in fast ripening
- Physiological effects of ethylene on plant tissue
leaf epinasty
epinastic curling of leaves on the right is due to flooded conditions, waterlogged soil results in anoxic roots, roots are unable to respire preventing the transport of water to the stem and leaves, all processes need to stop, ethylene is sent up the plant from the roots to cause curling, guard cells close and leaf surface is reduced reducing water loss and leaf surface capable of photosynthesis to reduce actions in the plant until conditions become more favourable.
flower senescence
chemical treatment in this case silver dip prevents the senescence of the flowers
Nowadays genetically modfiying flowers to be insensitive to ethylene or to not produce it is more common
root hair formation
ethylene increases root hair development (root hairs increase surface area) normally only some of the epidermal cells produce root hairs but under increased ethylene levels they are all converted to root hair producing cells. This was confirmed by genetically engineering plants without ethylene production in their roots resulting in no production of root hairs.
leaf abscission
ethylene triggers senescence – maintaining a green leaf requires a positive signal (auxin) at all times, this auxin tells the single cell layer that connects the leaf to the branch to prevent shedding. As the leaf ages auxin levels fall allowing ethylene to accumulate around the abscission point to cause programmed cell death – this means it only happens when the nutrients have been reabsorbed and leaf is ready to shed.
4.Genetic determination of ethylene signalling
The triple response in arabidopsis
Forward genetic screening – working from mutants to study what causes the phenotypic differences in their genomes. Growing long in the dark is a natural response that dicots use to reach the sunlight from underground.
Triple response only occurs in the dark
Addition of ethylene causes the ‘triple response’ short stem, thickening of stem and exaggeratio
n of the apical hook. Arabidopsis is a model organism but this strange growth response has been seen in many crop plants and other plant varieties also.
This response makes the stem stronger and protects the meristem to push through compacted soil.
The triple response phenotype (see photos)
1.INHIBITION OF HYPOCOTYL (STEM)
2.PROMOTION OF HYPOCOTYL THICKENING
3.EXAGGERATED TIGHTENING OF APICAL HOOK
Screen for ethylene insensitive mutants for Arabidopsis
forward genetic screening – finding mutants that don’t respond to ethylene – they will not show the triple response in the presence of heightened ethylene levels
Ethylene receptor action is based on the phenotype of receptor mutants - a mutation in one gene therefore inhibits the ethylene response pathway
Arabidopsis has 5 genes that encode the ethylene receptor
^ How can a mutation in one have a phenotype?
Normally other genes compensate?
NOT IN THIS CASE:
The reason we see a phenotype is because these receptors work differently to others.
* Ethylene inactivates the receptors, the receptors are active all the time in the absence of ethylene.
* When ethylene is present a response occurs but we know that ethylene binding inhibits the receptor
*This means the default state of a plant is to grow as if in the presence of ethylene
* Rather than switching things on in the presence of ethylene the pathway you are stopping the ethylene response pathway
* When ethylene binds it inhibits the receptors allowing the ethylene pathway to occur
SIMILARLY light does not switch processes on, processes are just prevented in the dark. A mutation causes plants to develop chloroplasts and open leaves in the dark which shows that it is the dark that is preventing it.
^This form of regulation allows fast response in plants – essential for sessile organisms
Auxin lecture:
1.Early experiments
2.The polar transport of auxin – auxin travels in one direction
3.The role of AUX1 and PIN1 proteins in auxin responses
4.The auxin receptor and regulation of auxin-regulated genes
5.Role of auxin in acid-induced growth
- Early experiments
Be aware that the basic historical experiments underpin modern research
The strawberry “fruit”: a swollen receptacle
Removing the strawberry achenes prevents growth
Adding auxin causes the strawberry to develop in the absence of achenes
Summary of early experiments in auxin:
- Darwin found that chopping off the top of the stem
prevents phototropism
(^responding to light and direction of light)
-Boysen-Jensen – mica insert (silicate – electric conductor) affects response, a jelly layer allows chemical conduction between the severed tip
- Paal in 1919 removed the tip of the stem and replaced it to one side causing growth curvation in the opposite direction even in unilateral light
- Went (1926) placed coleoptile stem tips on a gelatin sheet collecting the chemical passing through the gelatin.
He then applied this gelatin to one side of the growing cut stems with severed tips and he measured the angle the plants grew at (quantitative)
^ The more coleoptile tips used resulted in higher auxin concentration in the gelatin and stronger growth direction effects
Evidence that the lateral redistribution of auxin is stimulated by unidirectional light
Light does not degrade auxin instead auxin moves from a uniform distribution to one side
Dividing the coleoptile vertically with a mica sheet that cut through the tip all the way and divided the gel resulted in no difference in angle either side
Partially dividing the tip resulted in redistribution of auxin across the tip resulting in curving towards the light.
^Gus protein turns blue with a histochemical stain so you can see the auxin distribution by this blue colouration. Unidirectional light results in stronger colouration on the side with more auxin (shaded side)
- The polar transport of auxin – auxin travels in one direction
Donor–receiver block method for measuring polar auxin transport (see diagram)
radioactive auxin at the top and stem in regular orientation auxin moves down
When the stem is inverted and the radioactive auxin placed ontop auxin is not conducted
Peanuts stems grow upward (pos gravitrophic) once their flowers are fertilised they become negatively gravitrophic – the auxin must be changing polarity – resulting in the fertilised flowers digging into the ground before developing into ground nuts.
https://cdnsciencepub.com/doi/10.1139/b03-024#:~:text=The%20peanut%20gynophore%20is%20sensitive,take%20place%20throughout%20its%20development.
- The role of AUX1 and PIN1 proteins
A chemiosmotic model of polar auxin transport
-Typically cell wall is 5.4-5.8 pH (weakly acidic) auxins are weak acids so they are able to associate with protons in acidic conditions and become neutral to move passively through membranes
-This passive system is not enough to drive movement of auxing so permease cotransporters in the top of the cell drive the charged version through the membrane by proton motive force.
-In the cytosol protons are rarer so the majority of the auxin in the heart of the cell is in the charged form which cannot pass through the membrane transporters such as PIN1 use the hydrostatic difference to charge the movement.
- Asymmetric layout forces auxin downwards – polar transport
Auxin permease AUX1 is specifically expressed in a subset of tissues
- proteins need to be tagged e.g. with jellyfish derived
- fluorescent markers
These auxin proteins collect at the top of the cell
Whilst pin proteins are found at the bottom of the cell
- Auxin receptor and regulation of auxin-sensitive genes
Auxin makes the cell walls more plastic
*Acidification of the cell wall makes it more loose allowing cell growth
*Cell walls are already somewhat acidic (5.2-5.8 pH)
*To acidify cell wall further the cell must increase the number of protons in the cell wall
*To do this protons are pumped from the inside of the cell to the wall, this is achieved via proton pumps
*Which push against the gradient and require ATP
~1000 genes are upregulated by auxin but we are focusing on one key interaction:
Auxin binding to TIR1/AFB auxin receptors
1) Auxin response gene: a gene that encodes a protein
AuxRE - Auxin regulatory element a DNA motif upstream of coding regions switched on by auxin
ARF+ ARF dimer required to activate AuxRE in the absence of Auxin Aux/IAA binds and acts as a repressor preventing activation of AuxRE.
In the presence of auxin the repressor Aux/IAA is tagged to be degraded. When a protein is tagged with ubiquitine on specific lysine residues it is identified as a protein to be broken down.
2) Aux/IAA is sent for degradation in the proteasome and now ARF can form a dimer
3) once an ARF dimer is formed the transcription is activated
^Neg regulate the neg regulator to cause a positive response (double neg.) very common in plants
- Role of auxin in acid-induced growth
Auxin causes acidification of the cell wall allowing it to stretch so that the cell can grow
Current models for IAA-induced H+ extrusion
auxin stimulates proton atp-ase through transcription/translation of the H+ atpase
Auxin binds proteins between golgi and ER to assist in trafficking
Auxin stimulates the elongation of oat coleoptile sections
^ members of the grass family have a coleoptile – a tube of tissue protecting the new leaves
*Sections of this can be grown in a petri dish by adding auxin – growth is stimulated in only one direction.
*Protons being brought to the cell wall are likely to leak thus acidifying the water around the coleoptiles
*Whilst auxin itself is acidic only a small quantity is added and it is not a strong acid therefore increased acidity in the coleoptile plate after growing with auxin confirms the proton pump action
Kinetics of auxin-induced elongation and cell wall acidification in maize coleoptiles
^exponential growth is preceded by pH change = pH controls growth
*This is correlation how can we confirm causality?
*Adding a buffer that goes into the cell wall would prevent this acidification resulting in no rise in growth.
Gibberelins lecture:
1.Roles and synthesis of gibberelins
2.Molecular mechanism of gibberelin action
Background on gibberelins
Gibberelins act in a yes/ no fashion – shall we grow/or not
This is different to the activity of auxin which regulates how much growth occurs
Why do gibberelins prevent maximal growth? Maximal growth can only occur with very loose cell walls which puts a plant more at risk of biotic and abiotic factors thus reducing stress tolerance energy must be invested correctly (in a trade off.)
1.Roles and synthesis of gibberelins
The effect of exogenous GA1 on wild-type and dwarf (d1) maize
*plants will grow maximally in the ideal conditions and therefore cannot be further stimulated by gibberelin addition as seen in the normal plants.
* Dwarf plants grown in the same conditions lacking gibberelin function are unable to grow maximally
* Introducing gibberelins causes the dwarf plant to grow similarly to a normal one.
Cabbage can be induced to bolt and flower by applications of GA3
*In nature cabbage plants bolt and flower, domestic cabbage species have been bred to remain in a vegetative form better for eating – they have been bred not to respond to gibberelins.
*During the green revolution the aim was to reduce the amount of wasted effort e.g. modern wheat varieties grow shorter reducing energy wastage in straw production.
Gibberellin induces growth in Thompson’s seedless grapes
*nowadays most grapes are seedless, this was caused by spraying them with gibberelins and now is by genetic modification. These plants are reproduced by propogation (from cuttings)
Stem elongation corresponds closely to the level of GA1
* see diagram: le-2 has the smallest length between nodes – has a log difference in endogenous levels of gibberelins – far lower than wild type (LE)
*see diagram: identical genetically except for gibberelin oxidase gene being overexpressed – GA oxidase enzyme destroys gibberelins reducing natural GA levels.
These 4 plants have responded diffferently to the same genetic modification – why?
* Typically agrobacterium is used as a vector in genetic modification, or by shooting in gold/silver with the DNA sequence being added.
What you know in GM is the gene you are adding however it is inserted randomly into the genome and you can’t yet control that:
* where it lands in the DNA decides how active this added DNA is and whether it is impacted by other neighbouring genes. There are more accurate methods in other species but not yet available in higher plants.
^Variation in outcome is useful as this graded response allows you to select ideal plants
