How to Feed the World? Studies on the plant and abiotic stress interaction.

7/27/2019 How to Feed the World? Studies on the plant and abiotic stress interaction.

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Choon Kiat Lim

How to feed the world? Studies on the

plant and abiotic stress interaction


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What reason prompted me to pursue research in Plant Stress Tolerance?


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This provoked the following questions:-

Why there are still people living in such deplorable

situation, with basic rights for food being neglected?

What can I do as a future scientist?

What reason prompted me to pursue research in Plant Stress Tolerance?


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Impacts of environmental constraints on food security


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Therefore, understanding the mechanism of stress tolerance

in plants and strategy to develop stress tolerant crop are

essential to overcome a potential food crisis.


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Arabidops is thal iana, model in plant science research

The following advantages have madeArabidopsis a model

organism for many researchers in various fields of plant science:

Small genome (125 Mb) has been sequenced in the year 2000.

Extensive genetic and physical maps of all 5 chromosomes

A rapid life cycle (about 6 weeks from germination to mature

seed).

Prolific seed production and easy cultivation in restricted space.

Efficient transformation methods utilizingAgrobacterium

tumefaciens.

A large number of mutant lines and genomic resources many of

which are available from stock centers.

Multinational research community of academic, government and

industry laboratories.

Sou rce: TAIR


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What is ascorbate (AsA)?

Vitamin C antioxidant

An essential component of the

human diet

Essential for plant growth and

defence, regulated by the redoxstatus of ascorbate

Dowdle et al(2007)


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Ascorbate metabolism

**Apoplast: region in cell

wall & intercellular space.

**AsA: reduced form of

ascorbate

**DHA,dehydroascorbate:

oxidised form of

ascorbate


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Ascorbate oxidase (AO) an enigmatic enzyme in

search of a role

Image source: http://nobelprize.org/nobel_prizes/medicine/laureates/1937/szent-gyorgyi-bio.html?print=1


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AsA DHA + H2O

O2

Ascorbate oxidase (AO) an enigmatic enzyme in search of a role

Regulation ofapoplastic AsA level

Cell expansion, i.e. high AO activity in young / developing tissue

Oxygen management, i.e. low AO activity increases oxygen availability during

hypoxia (low oxygen).

Stress response, i.e. increased sensitivity to ozone inAO overexpression

tobacco

In Arabidopsis, AO is encoded by a small family of genes:

At4g39830 (AO1), At5g21105 (AO2) & At5g21100 (AO3)


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Ascorbate oxidase (AO) an enigmatic enzyme in search of a role

Genetic manipulation ofAO gene in tobacco and tomato showed that the effects of

AO in plant growth and development is small.

Contribution of apoplastic AsA (by AO) is in doubt:

ozone sensitive poplar and clover cultivars had higher total apoplastic AsA

content and greater AsA redox state than it respective resistant cultivars


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Environmental stress,

growth & development

AO

Apoplast (cell wall & intracellular space Symplast

ASA

DHAASA

DHA

or

Apoplastic AsA redox status

Research Questions

Plant Cell 1 Plant Cell 2

???

AsA levels


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Talk outlines:

The role of AO in leaf growth ofArabidopsis thaliana.

The role of AO during high light stress inArabidopsis thaliana.


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Phylogenetic

analyses of AO

Hi h AO i i i f d i i l i i f


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High AO activity is found in actively growing region ofA. thal iana:

Leaf expansion stops first at the tip,

and later at the base

Hi h AO ti it i f d i ti l i i f


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High AO activity is found in actively growing region ofA. thal iana:

Ch t i ti f AO T DNA i ti t t d l th


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Characterisation ofAOT-DNA insertion mutants under normal growth

conditions

1. Phenotype

2. AO activity

3. AsA level (whole leaf and apoplast)



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conditions



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conditions



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conditions

leaves

flowers

Whole leaf


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Whole leaf

Apoplast

Characterisation of AO T DNA insertion mutants in response to high light


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Characterisation ofAOT-DNA insertion mutants in response to high light

1. Phenotype

2. Anthocyanin concentration

3. AO activity

4. AsA level (whole leaf and apoplast)

Characterisation of AO T DNA insertion mutants in response to high light


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Characterisation ofAOT-DNA insertion mutants in response to high light

WT ao1 ao3 ao1ao3


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AO activity is increased during high light stress.

During high light stress, up regulation of the cytochrome b561 is believed to

regenerate more apoplastic AsA that can be used as a substrate for AO.

High light induced photooxidative stress. Reducing equivalents in chloroplasts

could be transported to the cytosol via malate-oxaloacetate shuttle to the

apoplast via plasma membrane bound MDHAR or cytochrome b561.

These reducing equivalents can be used by AO for the reduction of oxygen towater.


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Whole leaf

Apoplast

N h diff b d WT


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20% AO activity observed in ao3 & ao1ao3 mutants did not affect development

& stress response.

Complete suppression or over-expression is required to elucidate the function

of AO inArabidopsis thaliana.

Insertion mutants inAO2are not available.AO2is next toAO3 in the genome,

so double mutants would be difficult to make in any case. An RNAi approach is

being pursued with an attempt to silence allAO genes.

No phenotype difference between ao1, ao3, ao1ao3and WT

A tifi i l i RNA ( iRNA) h t il


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Artificial microRNA (amiRNA) approach to silence AOgenes

amiRNA hpRNAi

Source: Ossowski, S., Schwab, R., and Weigel, D. (2008).

Gene silencing in plants using artificial microRNAs and other small RNAs.

The Plant Journal 53, 674-690.


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amiR-AO

35S::AO3

Characterisation of amiR-AO & 35S::AO3 under normal growth conditions


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Characterisation ofamiR-AO& 35S::AO3under normal growth conditions

Characterisation of amiR-AO & 35S::AO3 under normal growth conditions


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Characterisation ofamiR-AO& 35S::AO3under normal growth conditions


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Whole leaf

Apoplast

Conclusion


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Conclusion

AO is highly conserved in higher plants. AO2 and AO3 ofA. thaliana showed

greater sequence identity than AO1.

High AO activity is found to be present in actively growing tissues of WT

A.thaliana during vegetative and reproductive growth stages.

Studies using T-DNA mutants showed that the effect of AO deficiency (10-20%

AO activity) in plant growth and development is small.

Conclusion


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Conclusion

The isolation ofamiR-AO plants showed that the lack of AO activity is not lethal

to plants. Three hypotheses are proposed for the basis of enhanced leaf growth

in the amiR-AO line:

1) the lack of AO could enhance the rate of OH-mediated non-enzymic cell

wall scission,

2) the lack of AO could increase auxin concentration in plants and

3) the lack of AO could cause the up-regulation of cell wall components in

order to compensate the abolition of AO.

Acknowledgements


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Acknowledgements

Supervisors: Nick Smirnoff, Ozgur Akman & Murray Grant

Post-Doc: Mike Page

Omics: Hannah Florance & Venura Perera

Plant pathology: Marta De Torres Zabala

Lab members in Mezzanine & Murray Grant Labs


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Thank you

How to Feed the World? Studies on the plant and abiotic stress interaction.

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