Lecture 3 - Abiotic resistance

Question 1

What naturally salt and drought resistanct plants can be exploited?

Answer 1

Craterostigma plantagineum (resurection plant)
Mesembryanthemum crystallinum (Ice plant)

Question 2

What are the features of the resurrection plant?

Answer 2

Dry out over a couple of weeks add wate and its fine

Question 3

What are the featues of the ice plant?

Answer 3

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

Question 4

What protective strategies can be used against salt and drought tolerence?

Answer 4

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

Question 5

Why is excessive salt bad?

Answer 5

Monovalent cations compete with nutret uptake to reduce groth 
Damaging to enzyme activity 
Membranes
Photosynthesis
Competes with K uptake

Question 6

What is a halophyte?

Answer 6

Salt loving

Ice plant

Question 7

What osmoprotectants are found in plants?

Answer 7

Glycine betaine (Spinach - salt stress)
Trehalose
Proline
Mannitol (Celery - salt)

Question 8

Hoow has accumulation of osmoprotectants conferrred resistnce to drough and sal stress in rice?

Answer 8

1. Introduced two enzymes (Trehalose phosphate synthase/Trehalose phosphate phosphotase) under a single ABA inducible promoter
2. Trehalose phosphate synthase converts the activated glucose molecules to trehalose-6-phosphate, then trehalose phosphate phosphotase converts this to trehalose
3. Increased expression of these enzymes led to a higher accumulation of trehalose
4. Treated for 4 weeks under 100mM of Nacl, transgenic lines compared to non transgenic with and without stress
5. Transgenic lines did as well as the WT without stress but better with stress

This is dependent on plant species

Question 9

What are the problems of attempting to increase the expression of something using biosynthetic enzymes?

Answer 9

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

Question 10

How was salt tolerance conferred by overexpression pf a vaculolar NA+/H+ antiport in arabidopsis?

Answer 10

Tolerance by sequestering Na into the vacuole (Na into vacuole in exchange for protons - vacuole acidic compartment, proportion of protons in vacuole driving force)

1. Looked aat the membrane transporters that sequester sodium ions of the plant
2. Idenitfied sodium proton antiporter system in the vacuolar membrane (osmoprotein)
3. Overexpressed this in arabidopsis and test plants under different levels of NaCl
4. Mutant was not effected but the WT was
5. Look at concentraytion of Na in the tissue
6. Transgenic plant accumulated more, but was excluded from the cytoplasm and taken into the vacuole

Question 11

How was salt tolerance acheived in tomato by the overexpressoin of a vacuolar Na/H antiporter from arabidopsis?

Answer 11

Vacuolar Na/H antiporter: AtNHX1 (arabidopsis)

1. Introduced under a constituative promoter and transgene introduced into tomato
2. Wt and mutant grown in 200mM NaCl/5mM NaCl
3. Transgenic plants showed dramatic increase in performance compared to the wild type
4. 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

Question 12

How was salt tolerance conferred to oil seed rape by over expression of the Na/H antiporter from arabidopsis?

Answer 12

Trnagenic plants, grown in 200mM NaCl, level of protein analysed in a western blot
Higher protein better survival

Question 13

How has salt and drought tolerance been conferred in arabidopsis by the overexpression of an H pump?

Answer 13

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

Question 14

How was SOS1 identified?

Answer 14

1. Mutant screen in arabidopsis, using EMS
2. Large mutant population treated for growth in the presence o NaCl, looking to identify mutations in genes necessary for stress tolerance
3. If growth in salt stress conditions was decreased compared to the WT then a gene was mutated necessary for salt stress tolerance
4. Identified SOS1 (salt overly sensitive 1) located in the plasma membrane
5. Recomplementation of the gene under the control of a constitutive promoter recover the WT phenotype of growth on 100mM Nacl agar

Question 15

Where is SOS1 expression most beneficial and why?

Answer 15

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

Question 16

What does the arabidopsis salt tolerant gene SOS1 encode?

Answer 16

putative Na/H antiporter

Question 17

What was the result of the overexpression of the SOS1 Na/H antiporter in arabidopisis?

Answer 17

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

Question 18

How can you attain expression of a gene in a specific tissue?

Answer 18

1. Need to identify specific enhancers that confer tissue specificity, can use enhancer trapping to get these control elements
2. Introduce construct into plants by agrobacterium mediated tranformation. Get integration of DNA flanked by left and right borders
3. Use a trunctated protein that is not active unless an enhancer element is close by.
4. Use basic promoter from a yeast system for the expression of GFP
5. 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.
6. develop hundreds different lines and identify lines where GFP expressed in the vascular tissue of the root system (stele)
7. 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
8. 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

Question 19

Following the expression of a sodium transporter specifically in root stele (enhancer trapping) how was salt tolerance increased?

Answer 19

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

Question 20

Why is it beneficial to have the tissue specific expression of a sodium transporter in root stele as opposed to all over the plant?

Answer 20

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

Question 21

Where has this tissue specific expression to decrease salt stress been found naturally?

Answer 21

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

Question 22

How was the wheat grain yeild on saline soils improved by an ancestral Na transporter gene?

Answer 22

1. Introgression of HKT1 gene (Nax2 locus) from triticum monocum into durum wheat
2. Compared original to the introgression line when plants were grown on different Na concentraions in the soil
3. Measured mean sodium concentration in soil
4. Found that Na accumulates in the leaves
5. 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
6. 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

Question 23

in the introgression of the HKT1 gene, where is it expressed and how do we know this

Answer 23

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

Question 24

How can salinity tolerance be conferred in crop plants?

Answer 24

Tissue tolerance in shoot
Ion exclusions at the root level
Osmoprotectant production
Stress signalling

Question 25

How can salinity tolerance be conferred in the leaves?

Answer 25

1. 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 2. get accumulation of compatible solutes and osmoprotectants

Question 26

How can salinity tolerance be conferred in the roots?

Answer 26

1. SOS expression is most useful in the roots. Good to be able to express it in different tissue 2. However may also export sodium into Xyleoplast (bad) but this can be coubnteraacted by HKT 3. Manipulate cation channels at the root epidermis however these are used for potassium as well cuold be problematic

Question 27

What is Thellungiella halophila?

Answer 27

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

Question 28

How do siRNAs regulate stress tolerance?

Answer 28

1. Intricate network of gene regulation involving RNAi 2. Upon salt stress (increased concentration of ROS) genes respond to the increase in ROS and SROS is transcribed 3. 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) 4. P5CDH produces p5C dehydrogenase which results in a low concentration of proline (osmoprotectant) 5. 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 6. This leads to increased stress tolerance

Question 29

How can siRNAs be exploited to increase stress tolerance?

Answer 29

1. Make use of the regulatory system by enhancing produce of small RNAs which then cleave the occurance of negative regulators e.g. P5CDH OR 2. 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

Question 30

How is ABA involved in cold acclimation?

Answer 30

- enhanced tolerance to freezing temperatures - increased levels of abscisic acid (ABA) - ABA treatment can replace cold acclimation

Question 31

What is the process of cold acclimation?

Answer 31

1. Low temperature increases ABA levels 2. 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

Question 32

How was eskimo1 identified in arabidopsis?

Answer 32

Eskimo1 mutants are constitutively freezing tolerant 1. Isolated mutants with a gain of function mutation 2. Tested a large number of mutants and one was found to be able to survive a sudden beneath 0 drop in temperature 3. 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

Question 33

What are the features of freezing tolerance in Eskimo1?

Answer 33

1. Does not depend on the expression of several cold regulated (COR) genes 2. Tested to see if the plants were already acclimated before cold treatment 3. In non acclimated plants Eskimo1 does not show expression of COR therefore it is not already acclimated 4. But eskimo1 didn't grow well

Question 34

How was chilling tolerance conferred in rice?

Answer 34

COLD1 confers chilling tolerance in rice 1. Japonica rice grows in regions with lower temperatures and is more tolerant to chilling wheras Indica grows in warmer regions 2. crossing and generating a NIL of indica containing the locus from japonica conferring cold resistance (COLD1 - identified by gene mapping) 3. When not treated the growth was similar to the WT 4. 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)

Question 35

What was the genomic difference in indica and japonic lines at the COLD1 locus? What is the protein?

Answer 35

1. Indica has Met/Thr at 187 of the protein 2. 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

Question 36

Why could the genetic difference conferred by COLD1 between indica and japonic cultivars not have been developed in a mutant screen with EMS?

Answer 36

1. Non tolerant line has a T at position 187 and the tolerant line has a A. This is not possible by EMS 2. 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

Question 37

What signalling pathways are present for cold acclimation?

Answer 37

ABA through... 1. TF: CBF1 - COR gene expression 2. TF: CBF3 - leads to COR gene expression and proline accumulation 3. ESK1 leads to proline accumulation 4. COR and proline independent pathways

Question 38

What is the result of upregulation of COR genes, proline accumulation and Anti-freeze proteins?

Answer 38

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

Question 39

What is the result of an overexpression of CBF1?

Answer 39

Confers drought and freezing tolerance (TF in ABA COR gene signalling) Increase in CBF1 in brassica increases drought and freezing tolerance

Question 40

What is the result of the constitutive overexpression of CBF3/DREB1A?

Answer 40

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.

Question 41

Is the inducible expression of CFB3 and ABF3 for stress tolerance possible in crops?

Answer 41

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)

Question 42

How was this same idea transferred to Maize?

Answer 42

1. Characterise genes in arabidopsis 2. Identify ortholgs in crop plants 3. Found specific TF in arabidopsis that when overexpressed conferred drought tolerance (AtNF-YB1) in arabidopsis 4. 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

Question 43

How was drought tolerance conferred in Rice?

Answer 43

DRO1 1. 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) 2. Some regions where rice is grown have deeper roots to reach water, these varients were identified 3. Produced an introgression line with DRO1 which was shown to increase the gravitropism response. 4. In the WT flat root the roots grow at a small angle down but in the isogenic line the angle is steeper 5. Studied the response to gravity by growing on a plate, turned around, and the gravitropic response of the isogenic line was quicker 6. Tested under a lot of different conditions at different levels of drought and soil moisture at different depths beneath the surface 7. 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

Question 44

Give an example of important metal plant toxicity

Answer 44

Aluminum | 40% arable land has Al problems

Question 45

What are the mechanisms of Al-resistant plants?

Answer 45

1. Excretion of organic acids (malate, citrate) to form a complex with Al (once in complex it is not easily taken up) 2. 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

Question 46

Give an example of using the excretion of organic acids to decrease Al toxicity

Answer 46

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

Question 47

How has high level aluminum tolerance been shown in barley?

Answer 47

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

Question 48

What strategies can be used to increase stress tolerance?

Answer 48

1. Osmoprotectants 2. Ion transporters/channels to reduce salt stress 3. Transcriptional activators of stress tolerance 4. Potential use of siRNAs 5. Reducing metal toxicity by complex formation with organic acids 6. Successful identification of QTLs involved in stress tolerance

Question 49

List some of the abiotic stressors found in plants

Answer 49

``` Intense light Herbicides Ozone Heat Chilling Freezing Drought Salinity Flooding Heavy metals ```