Discovering the genetic basis for plant freezing tolerance Flashcards
Plant freezing damage, tolerance and cold acclimation
Freezing damage:
*Cellular dehydration
*Membrane damage
^ Mostly consequence of ice forming
(Protein misfolding can also be caused by cold itself)
* Freezing stress causes desiccation stress and membrane damage
- extracellulary ice forms draws water out of the cell resulting in desiccation (aka drought) stress
^ during thawing ice surrounding returns to liquid form and water floods in which can cause membrane damage due to fast expansion of the cytoplasm
We can quantify freezing damage by testing the leakiness of cells (% electrolyte leakage)
As temperatures drop below 0°C, ice formation is initiated in the intercellular spaces due to the extracellular fluid having a higher freezing point (lower solute concentration) than the intracellular fluid. So the first place water freezes is in the cell wall. This leads to water being drawn out of the cell cytosol (symplast) into the cell wall (apoplast) due to the enormous water potential gradient. Because the chemical potential of ice is less than that of liquid water at a given temperature, the formation of extracellular ice results in a drop in water potential outside the cell. (No “liquid water” outside and lots inside.)
Consequently, there is movement of unfrozen water down the chemical potential gradient from inside the cell to the intercellular spaces.
Arabidopsis thaliana: the model plant system we use to learn about freezing tolerance
Model systems are very important in biology. They are organisms for study, that represent other similar organisms but are often simpler and easier to use. Arabidopsis is chosen as its genome is smaller than many other plants so it is simpler to study; it is also small and easy to grow quickly. Luckily Arabidopsis is one of the species that CAN tolerate freezing temperatures so it is well worth using for our question.
Quantifying freezing tolerance using an Electrolyte Leakage (EL) assay
To conclude how much damage has been incurred
Significantly damaged cells will release more electrolytes as cells are more leaky
1)Freezing and thawing a leaf sample then quantifying the amount of electrolytes leaked
2) Freezing a sample of the same leaf at –80 degrees c to cause all electrolytes to leak out
3) comparing the total electrolyte quantity (from part 2) with the amount lost from regular freezing temperature (from part 1) allows us to quantify how tolerant a plant is to freezing
(by how leaky freezing makes the cells)
Cold acclimation can improve plant tolerance to freezing conditions
Temperate plants tend to be able to do this whereas tropical and sub-tropical plants cannot.
Cold acclimation is improving freezing tolerance through being exposed to a period of cold but not freezing temperatures.
Certain proteins accumulate during cold acclimation, changes in physiology and cell membranes result from a change in gene expression.
How cold acclimation combats the causes of freezing damage
Increased levels of compatible solutes – more difficult for water to get sucked out of the cell during the dehydration phase, by increasing the expression of genes that make enzymes to make those compatible solutes
Change in lipid composition - making membranes more robust and less likely to get damaged at cold temperatures/ during rehydration less likely to burs
Cold acclimation involves widespread changes in gene expression
*Few of these genes have a huge impact individually but collectively they do.
*Need to find the regulators that switch them all on
Many Cold-On Regulated (COR) genes share a common promoter motif
Mike Thomashow’s lab showed that CBF transcription factors bind to CRT motif switching on COR transcription
Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit - PubMed (nih.gov)
CBF1 genes are rapidly induced in Response to low temperature BEFORE the COR genes
No transcripts of CBF before cold, increase rapidly over first 3 hours, this correlates with the expression of COR increasing after this.
Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.
(Also from the Thomashow group). Shows expression of the three CBF genes is cold- inducible and precedes the expression of presumed target genes but doesn’t show they are linked.
but are they the cause of the COR gene being expressed?
Yes! CBF Transcription factors act as “Master Switches” to activate the cold acclimation response
CBF Transcription factors act as “Master Switches” to activate the cold acclimation response
See photos from lecture:
Proof that CBFs switch on many target COR genes and thus act as a master switch for cold acclimation. Plants behave as if cold acclimated even if they have never seen the cold. This works if any of the three CBF genes are overexpressed.
CBF1 overexpressor plants don’t require acclimatisation
This mechanism has been seen in many other plants including crops (not limited to Arabidopsis)
Mutant screens help identify important tolerance genes
How do we find out what makes things work?
A key approach in experimental science is to intervene in a system and see what the consequences of your intervention are. One was you can intervene is to eliminate a component of the system and see if it still works or is affected. If it is affected by the change you make you can deduce that the change you made caused the effect you saw and then you can link the function of the thing you changed (or eliminated) with the effect you observe.
How can we identify the components that make organisms work?
introducing a mutation to disrupt DNA to affect the protein it codes – like removing parts from a functioning car this will impact the functionality of the organism
^ Every protein in an organism is encoded by a gene. So if we want to know what the protein does, we need to see what happens when we remove it. We can do this by preventing the gene from expressing the protein, or “knocking the gene out”.
Mutant screens help identify important tolerance genes: Two ways to study mutants: Forward and reverse genetics
Reverse genetics: is starting with DNA, making a change to it and seeing what happens to its phenotype it is quick but only really useful if you think you have already identified the gene which is important – a biased approach relying on assumption
Forward genetics: starts with a phenotype of interest and goes back to identify the cause, the genetic mutation that resulted in the phenotype. This takes much longer than reverse genetics but has many advantages e.g. is completely unbiased leading to the identification of genes that were not previously considered.
(^this lecture will focus on forward genetics)
Introducing mistakes deliberately: Creating mutants
*Ethyl methane sulphonate (EMS ) is a commonly used mutagen
*EMS makes single base changes (SNPs)
Dangerous process normally researchers send seeds to specialists to carry this out
We have to introduce enough random mutations so that we have at least one individual with an error in every gene. In this case, the individuals are seeds, each of which will grow up to produce a plant with a small genetic difference. We need thousands of mutagenised seeds so we can ensure that there is at least one seed that has a mutation in every gene of the genome.
From the mutant plants we can do a forward genetic screen (FGS)
We have now made our thousands of mutant seeds and we are ready to discover which one of them will produce a plant that is different in a way that is interesting to us as experimenter.
We have to find the one plant that is different amongst a sea of others that all look normal.
1) Grow thousands of individuals each with unique genetic differences
2) Find the individual(s) that show a phenotype of interest
3) Identify the gene that was altered to get that phenotype
4) You now know what that gene (and its protein) does
To find the plants that are different: You can perform a genetic stream for plants with reduced/increased stress tolerance
see photos of a salt tolerance test:
^left all plants watered with a small amount of salt that wouldn’t affect wild type to ID salt sensitive mutants
(* salt sensitive mutants are visibly stunted)
^right all plants watered with high salt level to identify salt tolerant mutants (* only salt tolerant mutants survive)
In each case we are looking here for a plant that shows different sensitivity to the stress test. Its much easier to find the high tolerance mutants than the low tolerance ones.
Using sensitive-to-freezing (sfr) mutants to understand what is important in cold acclimation and freezing tolerance.
What genes are required for freezing tolerance: a forward genetic approach
^ Found that genes affecting sensitivity in freezing related to: Chloroplast membrane, wax synthesis, sugar accumulation, cell wall structure and CBF action – losing these genes results in plants losing tolerance
http://www.plantphysiol.org/content/111/4/1011
long Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Work ongoing in Durham now to assign genes to each of the sfr mutations. Only one of these mutants has any connection with CBF controlled gene expression (that’s sfr6).
SFR2 is involved in protecting the chloroplast during freezing, SFR3 increases cuticular wax production and SFR4 plays a role in accumulation of sugars during cold acclimation.
sfr6: a cold acclimation mutant. Gene expression.
Sfr6 is a high sensitivity mutant
Arabidopsis sensitive-to-freezing-6 (sfr6) is defective in COR (COLD) gene regulation
sfr6 responds weakly to cold stress – had an issue with CBF activation
Knight used a genetic approach to understand what goes on during cold acclimation and studied a number of Gary’s mutants. This is sensitive to freezing 6, an Arabidopsis mutant that cannot cold acclimate. We discovered its expression of cold inducible genes during the acclimation process is very poor. The mutant doesn’t respond to the opportunity to cold acclimate and it doesn’t upregulate well studied cold inducible genes like KIN2 when it gets colder. Showed these genes are likely to be important for cold acclimation.
Many other cold-responsive genes are affected by loss of SFR6
Microarray showed lots of genes (dozens) were affected in sfr6 mutants. We looked at cold regulated genes that were mis regulated in sfr6. Not all of the cold genes were mis reg, but those that were had a high incidence of a couple of promoter motifs; this was the top one. (Make CBF fly on and the arrow get thicker). The TF for the CRT/DRE is known.
SFR6 is MED16, a subunit of the Mediator transcriptional co-activator complex
MED16 (SFR6) shares control of COR gene expression with MED2 and MED14 but not others.
Freezing tolerance is impaired in med2 and med14 mutants, like med16 (sfr6)
none of the med2 and med14 mutants survive freezing cannot switch on COR genes as they lack the activation complex
SFR6 (MED16) mechanism: Model for RNA Polymerase (Pol II) recruitment to CBF-regulated genes
damaged tail results in a loss of connection so no recruitment of complex and no effective transcription
In summary: SFR6 is essential for acclimation-related freezing tolerance because helps bring the transcriptional machinery to COLD gene promoters so they can be expressed.
sfr8: a basal freezing tolerance mutant. Cell wall.
Sfr8 has a Mur 1 mutation resulting in cell wall issues – citing that cell wall is related to freezing protection
SFR8/MUR1 is a GDP-D-mannose-4,6-dehydratase: catalyses fucose synthesis
sfr8 / mur1 mutants cannot produce fucose. Fucose is used in fucosylation of other molecules.
Lack of fucosylation means that biological molecules are not connected properly.
Which cell wall fucosylation event is important?
see diagram
*Pectins: rhamnogalacturonans RGI and RGII (form a gel)
*xyloglucan (a hemicellulose)
*Arabinogalactan proteins (AGPs)
RGll was chosen for study as it has been connected to cell wall issues before
RG-II pectin can exist as monomer or dimer - in normal plants it is usually dimerised
In normal plants 95% is dimerised in the sfr8 mutant only 50% are dimers
This seems to affect freezing tolerance but we are not sure why
RGII dimerisation is reduced in sfr8 mutant
Restoring dimerisation of RG-II pectin improves freezing tolerance in sfr8
Connecting cross-link formation to freezing tolerance.
Boron is required to connect the dimers:
Watering plants with boron infused water can cause dimerisation in the absence of fucosylation this resulted in an improvement of freezing tolerance in sfr8 mutants showing that pectin cross-linking is necessary for freezing tolerance.
The connected diagram provides suggestions for the causes of this.
Using multi-disciplinary approaches to learn about plant physiology.
*Crosslinking between RGII pectin chains in the cell wall is reduced in sfr8 mutants.
*This affects physical properties of the cell wall.
*Are these properties important for resisting damage or limiting ice growth?
sfr8 mutant shows greater cell wall porosity
When cell plasma membrane is stained with a fluorescent marker and a quenching molecule with the ability to turn off the fluorescence is applied. The quencher is a large molecule so if flourescence is quenched it means that the cell wall has large pores, a poorer quenching level would suggest a less porous cell wall.
As seen the wildtype requires higher quenching conc. than in the sfr8 mutant.
Makes sense as sfr8 has fewer cross-links therefore larger holes.
Could cell wall porosity and strength impact upon freezing tolerance?
