Lecture 3 - Abiotic resistance Flashcards
What naturally salt and drought resistanct plants can be exploited?
Craterostigma plantagineum (resurection plant) Mesembryanthemum crystallinum (Ice plant)
What are the features of the resurrection plant?
Dry out over a couple of weeks add wate and its fine
What are the featues of the ice plant?
Crystals for on the surface of the shoots made up of sodium chloride
Plant can excrette excessive levels of salf
Doesn’t accumulate in tissue
What protective strategies can be used against salt and drought tolerence?
Reduced transpiration (stomato allow the uptake o Co2, water evaporates, transpiration stream allows uptake of water from the soil and salts and nurient - closing by the addition of ABA reduces water loss but not forever)
Accumulation of non toxic osmolytes (osmo protectants)
Sequestration of salt outside the cytoplasm
Why is excessive salt bad?
Monovalent cations compete with nutret uptake to reduce groth Damaging to enzyme activity Membranes Photosynthesis Competes with K uptake
What is a halophyte?
Salt loving
Ice plant
What osmoprotectants are found in plants?
Glycine betaine (Spinach - salt stress)
Trehalose
Proline
Mannitol (Celery - salt)
Hoow has accumulation of osmoprotectants conferrred resistnce to drough and sal stress in rice?
- Introduced two enzymes (Trehalose phosphate synthase/Trehalose phosphate phosphotase) under a single ABA inducible promoter
- Trehalose phosphate synthase converts the activated glucose molecules to trehalose-6-phosphate, then trehalose phosphate phosphotase converts this to trehalose
- Increased expression of these enzymes led to a higher accumulation of trehalose
- Treated for 4 weeks under 100mM of Nacl, transgenic lines compared to non transgenic with and without stress
- Transgenic lines did as well as the WT without stress but better with stress
This is dependent on plant species
What are the problems of attempting to increase the expression of something using biosynthetic enzymes?
Dont know what counteractions are present
Trehalose is degraded into trehalase (two gluscose molecules)
May have to interfere with turnover as well as increase expression
How was salt tolerance conferred by overexpression pf a vaculolar NA+/H+ antiport in arabidopsis?
Tolerance by sequestering Na into the vacuole (Na into vacuole in exchange for protons - vacuole acidic compartment, proportion of protons in vacuole driving force)
- Looked aat the membrane transporters that sequester sodium ions of the plant
- Idenitfied sodium proton antiporter system in the vacuolar membrane (osmoprotein)
- Overexpressed this in arabidopsis and test plants under different levels of NaCl
- Mutant was not effected but the WT was
- Look at concentraytion of Na in the tissue
- Transgenic plant accumulated more, but was excluded from the cytoplasm and taken into the vacuole
How was salt tolerance acheived in tomato by the overexpressoin of a vacuolar Na/H antiporter from arabidopsis?
Vacuolar Na/H antiporter: AtNHX1 (arabidopsis)
- Introduced under a constituative promoter and transgene introduced into tomato
- Wt and mutant grown in 200mM NaCl/5mM NaCl
- Transgenic plants showed dramatic increase in performance compared to the wild type
- Western blotting on plama membrane/ER/ tonoplast showed increase in protein in the vacuolar membrane. Done by using markers known to be in tonoplast and by subfractioning the different compartments saw high level of accumulation in the tonoplast.
Transgenic tomato were sightly smaller, protein accumulated salt in the shoot tissue not in the fruit
How was salt tolerance conferred to oil seed rape by over expression of the Na/H antiporter from arabidopsis?
Trnagenic plants, grown in 200mM NaCl, level of protein analysed in a western blot
Higher protein better survival
How has salt and drought tolerance been conferred in arabidopsis by the overexpression of an H pump?
AVP1 H pump
Action: cleaves phosphotase (high energy bond) into 2 inorganic phosphates
Energy is used to pump protons into the vacuole
Making this pump more efficient supports the proton gradient utilised by the Na/H antiporter, therefore greater accumulation of Na in vacuole
1/ Overexpressed AVP1 pump in vacole
2. Immunolocalisation of the AVP1 protein in western blot
3. Mutant showed salt tolerance unpon watering with 250 mM NaCl
4. Also showed drought tolerance (not watered for 10 days, watered, survival)
Could combine the Na/H antiporter and pump to increase efficiency
How was SOS1 identified?
- Mutant screen in arabidopsis, using EMS
- Large mutant population treated for growth in the presence o NaCl, looking to identify mutations in genes necessary for stress tolerance
- If growth in salt stress conditions was decreased compared to the WT then a gene was mutated necessary for salt stress tolerance
- Identified SOS1 (salt overly sensitive 1) located in the plasma membrane
- Recomplementation of the gene under the control of a constitutive promoter recover the WT phenotype of growth on 100mM Nacl agar
Where is SOS1 expression most beneficial and why?
In the root epidermis
Can’t be expressed everywhere because it woul be counteracted
Potential to manupulate ion channels in the epidermisfor K transport as have a similar structure to Na
What does the arabidopsis salt tolerant gene SOS1 encode?
putative Na/H antiporter
What was the result of the overexpression of the SOS1 Na/H antiporter in arabidopisis?
Overexpression of SOS1 increased salt tolerance
Following growth upon salt treatment mutant did better than WT
Look at the accumulation of Na in tissue
As concentration increases in the WT, in the independent transgenic lines see a reduction in the acculuation of Na
Salt stress increases mRNA stability (northern blot analysis) of SOS1
Shown as steady state level of SOS1 transcript when under 35s constitutive promoter but RNA is degraded without salt
Beneficial: Using energy to produce and degrade RNA is much less than the energy required for protein production if it is not needed
This is an inducible system
How can you attain expression of a gene in a specific tissue?
- Need to identify specific enhancers that confer tissue specificity, can use enhancer trapping to get these control elements
- Introduce construct into plants by agrobacterium mediated tranformation. Get integration of DNA flanked by left and right borders
- Use a trunctated protein that is not active unless an enhancer element is close by.
- Use basic promoter from a yeast system for the expression of GFP
- If a transcription factor binds promoter get GFP expression by the activity of an upstream activator sequence. This occurs when the tDNA has integrated into the host chromosome where there are tissue specific enhancers.
- develop hundreds different lines and identify lines where GFP expressed in the vascular tissue of the root system (stele)
- Use this line to cross to a line with the gene of choice (Na transporter) using a UAS sequence linked to the transporter and selectible marker
- Transcription factor in the first system acts in trans on any gene controlled by UAS (transactivator system) therefore where GFP expressed get expression of the transporter
Following the expression of a sodium transporter specifically in root stele (enhancer trapping) how was salt tolerance increased?
Salt tolerance increased as the sodium transporter in the root stele reduced shoot Na accumulation.
HKT line had the sodium transporter highly expressed in the vascular tissue
Transgenic plants were more tolerant then WT under high concentration of salt
% tolerance = % dry weight under salt condtions compared to the WT
Why is it beneficial to have the tissue specific expression of a sodium transporter in root stele as opposed to all over the plant?
Don’t want to express proteins everywhere if don’t need to, exess energy use
Use:
Inducible expression, Tissue specific expression
Otherwise have detrimental effects without stress
Where has this tissue specific expression to decrease salt stress been found naturally?
Found naturally in ancestral wheat accessions
Tissue specific expression of transporter in cells adjacent to the Xylem in roots , bringing Na from the transpiration stream into the shoot as this is less detrimental in the root than in the shoot
How was the wheat grain yeild on saline soils improved by an ancestral Na transporter gene?
- Introgression of HKT1 gene (Nax2 locus) from triticum monocum into durum wheat
- Compared original to the introgression line when plants were grown on different Na concentraions in the soil
- Measured mean sodium concentration in soil
- Found that Na accumulates in the leaves
- In the original Na in the leaves far higher than the introgressed line where the concentration was far lower, showed better growth on high salt soils
- But slightly worse growth on WT soils
Classical breeding can be sped up by molecular techniques
1. Gene sequencing and use genotyping to see if the introgression has been aquired
in the introgression of the HKT1 gene, where is it expressed and how do we know this
HKT1 gene expressed in the vascular tissue
In situ PCR (PCR combined with in situ hybridisation and staining)
Amplify the cDNA in fixed tissue and stain it
1. In the introgressed line got tissue specific staining near xylem
2. Used 16s RNA expresssion as control (seen everywhere)
3. without reverse transcriptase before staining get background stain
High expression in roots not shoots in response to salt stress
How can salinity tolerance be conferred in crop plants?
Tissue tolerance in shoot
Ion exclusions at the root level
Osmoprotectant production
Stress signalling
