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
How can salinity tolerance be conferred in the leaves?
- different types of ATPases pump protons into the vacuole, this gradient can be used to exchange protons for things into the vacuole where they won’t do harm
- get accumulation of compatible solutes and osmoprotectants
How can salinity tolerance be conferred in the roots?
- SOS expression is most useful in the roots. Good to be able to express it in different tissue
- However may also export sodium into Xyleoplast (bad) but this can be coubnteraacted by HKT
- Manipulate cation channels at the root epidermis however these are used for potassium as well cuold be problematic
What is Thellungiella halophila?
Thellungiella halophilia is a halophyic relative of arabidopsis
Looked at the gene sets to determine genes controlling salt tolerance
But genes were similar
Regulation is different
How do siRNAs regulate stress tolerance?
- Intricate network of gene regulation involving RNAi
- Upon salt stress (increased concentration of ROS) genes respond to the increase in ROS and SROS is transcribed
- The gene for the mRNA that is produced has a 3’ end which overlaps with a gene located on the lower strand of the chromosome which is constitutively expressed and involved in proline metabolism (P5CDH)
- P5CDH produces p5C dehydrogenase which results in a low concentration of proline (osmoprotectant)
- The system works by producing hybrid dsRNA which is processed by the silencing system into small pieces (20nt) which degrades RNA leading to decreased p5c dehydrogenase and increased proline
- This leads to increased stress tolerance
How can siRNAs be exploited to increase stress tolerance?
- Make use of the regulatory system by enhancing produce of small RNAs which then cleave the occurance of negative regulators e.g. P5CDH
OR - Positive regulators may be negatively regulated by small RNAs, downregulate negative small RNAs to get more positive regulators
If identify regulatory systems can manipulate them to improve stress tolerance
How is ABA involved in cold acclimation?
- enhanced tolerance to freezing temperatures
- increased levels of abscisic acid (ABA)
- ABA treatment can replace cold acclimation
What is the process of cold acclimation?
- Low temperature increases ABA levels
- Cascade initiated including secondary messengers (cADP ribose, cyosolic free Ca) to lead to the expression of COR genes (cold-regulated genes)
However there are also ABA independent pathways
How was eskimo1 identified in arabidopsis?
Eskimo1 mutants are constitutively freezing tolerant
- Isolated mutants with a gain of function mutation
- Tested a large number of mutants and one was found to be able to survive a sudden beneath 0 drop in temperature
- Mutant showed difference in the ability to tolerate sudden cold. Even if we allowed for acclimation, (treating plants for two days at 4 degrees) the wt can also tolerate low temperatures but the mutant can do better
What are the features of freezing tolerance in Eskimo1?
- Does not depend on the expression of several cold regulated (COR) genes
- Tested to see if the plants were already acclimated before cold treatment
- In non acclimated plants Eskimo1 does not show expression of COR therefore it is not already acclimated
- But eskimo1 didn’t grow well
How was chilling tolerance conferred in rice?
COLD1 confers chilling tolerance in rice
- Japonica rice grows in regions with lower temperatures and is more tolerant to chilling wheras Indica grows in warmer regions
- crossing and generating a NIL of indica containing the locus from japonica conferring cold resistance (COLD1 - identified by gene mapping)
- When not treated the growth was similar to the WT
- Following cold treatment (24 days after) the NIL did much better
Shows the possibility of using mapping technlogy to identify a gene varient that controls a specific trait
COLD1: single aa change (SNP)
What was the genomic difference in indica and japonic lines at the COLD1 locus? What is the protein?
- Indica has Met/Thr at 187 of the protein
- Cold tolerance has Lys at 187 of the protein
Single amino acid change confers cold tolerance
Transmembrane protein interacting with some G proteins in a signalling cascade to regulate the activity of calcium channels so that the calcium concentration in the cell is increased
Why could the genetic difference conferred by COLD1 between indica and japonic cultivars not have been developed in a mutant screen with EMS?
- Non tolerant line has a T at position 187 and the tolerant line has a A. This is not possible by EMS
- EMS creates a mutation from C/G to A/T (with G moving to A, and the C to a T). Cannot create a T to A change.
Therefore must look for natural variation and make use of it
What signalling pathways are present for cold acclimation?
ABA through…
- TF: CBF1 - COR gene expression
- TF: CBF3 - leads to COR gene expression and proline accumulation
- ESK1 leads to proline accumulation
- COR and proline independent pathways
What is the result of upregulation of COR genes, proline accumulation and Anti-freeze proteins?
COR genes
-enhances freezing tolerance
Proline accumulation
-enhances freezing tolerance (osmoprotectant)
Anti-freeze proteins
-prevent formation of large ice crystals when sudden drop in temperature, instead smaller with rounded edges
What is the result of an overexpression of CBF1?
Confers drought and freezing tolerance (TF in ABA COR gene signalling)
Increase in CBF1 in brassica increases drought and freezing tolerance
What is the result of the constitutive overexpression of CBF3/DREB1A?
In the absence of stress the plants are smaller
Inducible overexpression controlled by a cold inducible promoter confers drought, salt and freezing tolerance by controlling expression of the COR genes and the mutants have the same growth as the WT under normal conditions.
Is the inducible expression of CFB3 and ABF3 for stress tolerance possible in crops?
Transgenic approach leads to problems (unstable phenotype) wheras classical breeding generates phenotypes that are more stable
Constitutive overexpression of Arabidopsis CBF3 and ABF3 in rice confers stress tolerance without compromising on growth.
1. Arabidopsis CBF3 and ABF3 introduced into rice
2. Under drought conditions the WT suffered wheras the transformed plants recovered (following the constitutive overexpression of both tTF)
How was this same idea transferred to Maize?
- Characterise genes in arabidopsis
- Identify ortholgs in crop plants
- Found specific TF in arabidopsis that when overexpressed conferred drought tolerance (AtNF-YB1) in arabidopsis
- When the maize ortholog (ZmNF-YB2) was overexpressed in the glasshouse and field studies got a dramatic increase in yeild in two transgenic lines compared to the control lines
By interfering with the expression of a single central regulator can acheve dramatic changes in the crop plant
How was drought tolerance conferred in Rice?
DRO1
- Plants with a flat root system are not tolerant to drought conditions (if surface of the soil dries out it can’t cope) - upland rice (IR64)
- Some regions where rice is grown have deeper roots to reach water, these varients were identified
- Produced an introgression line with DRO1 which was shown to increase the gravitropism response.
- In the WT flat root the roots grow at a small angle down but in the isogenic line the angle is steeper
- Studied the response to gravity by growing on a plate, turned around, and the gravitropic response of the isogenic line was quicker
- Tested under a lot of different conditions at different levels of drought and soil moisture at different depths beneath the surface
- NIL performed better in filled grain and grain weight compared to the WT
Critisisms: Overlapping error bars. Check error bars. If S.E overlap then they are not significanly different as it is the standard deviation of the mean
But used ANOVA stats test so okay
Give an example of important metal plant toxicity
Aluminum
40% arable land has Al problems
What are the mechanisms of Al-resistant plants?
- Excretion of organic acids (malate, citrate) to form a complex with Al (once in complex it is not easily taken up)
- Increase in rhizosphere pH to form Al hydroxides (low solubility)
However plants need to acidify the rhizosphere to for nutrient uptake systems, therefore the excretion of organic acids is more promising
Give an example of using the excretion of organic acids to decrease Al toxicity
Complex formation of Aluminum with oragnic acids alleviates Al txicity
1. Wheat seedlings treated with AlCl and root elongation was reduced
2. If add different organic acids at same time get less bad results from high Al
Can visualise Al uptake by staining in the root tips (sensitive to Al concentrations.)
Hydranga can form natural complexes with Al and organic acids and forms a blue colour in acidic soils
How has high level aluminum tolerance been shown in barley?
Malate transporter ALMT1 confers high level aluminum tolerance to barley
1. Malate transporter from wheat (tolerant to AI, barley not. ET8 Al-tolerant line )
2. Take transporter and create barley transgenic lines
3. See a dramatic difference in barley tolerance at increasing concentrations of Aluminum
4. Three different transgenic lines with much bettter root growth, similar to the donor line from wheat
5. Also grew well on acidic soils
Electron microscopy shows what happens when Al too high - deformation of root tips
Staining reaction shows blue staining with a higher accumulation of Al in root tips
What strategies can be used to increase stress tolerance?
- Osmoprotectants
- Ion transporters/channels to reduce salt stress
- Transcriptional activators of stress tolerance
- Potential use of siRNAs
- Reducing metal toxicity by complex formation with organic acids
- Successful identification of QTLs involved in stress tolerance
List some of the abiotic stressors found in plants
Intense light Herbicides Ozone Heat Chilling Freezing Drought Salinity Flooding Heavy metals
