Plant ThermotolerancePlant Thermotolerance- - hot1-3 hot1-3 (HSP101)(HSP101)

HORT 301HORT 3019/30/20089/30/2008

Your goal:Discover New Genes that function for Thermotolerance

Choose one of the following:

Reverse genetic approach

Forward genetic approach

Reverse genetic approach for thermotolerancemutant plants are identified based on screening a large population harboring random mutations for a phenotypic change

1. Select a gene or genes - Complete genome sequences provide the information of genes or

gene family

Select tomato HSF1a

Baniwal et al., 2004 J. Biosci.

2. Generate mutants or overexpression lines 3. Test hypothesized responses (Thermotolerance)

ControlOver-expression

Co-suppression

Mishra et al., 2002 Genes and Development

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Forward genetic approach for thermotolerancemutant plants are identified based on screening a large population harboring random mutations for a phenotypic change

1. Select a biological process - Acquired thermotolerance for Dark-induced hypocotyl growth

24C 45C 2 hr 38C 1.5hr45C 2 hr

Chan Yul’s experiment

2. Generate a random and highly redundant mutant population Ethyl methanesulfonate – chemical modification of nucelotides C T

Forward genetic approach for thermotolerance

To achieve saturation of EMS mutagenesis in Arabidopsis, at least 125,000 M1 lines should be generated (Jander et al., 2003; Kim et al., 2006)

3. Screen a large number of mutagenized M2 plants 17,000 M2 seeds were screened (from estimated 2,100 M1 parents)

Sow 17,000 seeds on the medium in the square plates (As we did last week!)

Mark the seedlings that do not show acquired thermotolerance

Rescue the seedlings

Forward genetic approach for thermotolerance

Hong and Vierling, 2000 PNAS

4. Map and clone the mutation

Jander et al., 2002 Plant Physiol

5. Confirm by Complementation test

Hong and Vierling, 2000 PNAS

So, HSP101 gene functions for acquired thermotolerance

6. Confirm by another allele (hot1-3)

Hong and Vierling, 2000 PNAS

HSP101 gene (At1g74310)

hot1-3 is a T-DNA insertion mutant that eliminates HSP expression

HSP101HSP101

Function of HSP101: Molecular chaperone; ATP-dependent dissociation of protein aggregates

Baniwal et al., 2004 J. Biosci.

Katiyar-Agarwal et al, 2003 Plant Mol. Biol.

Can we generate Heat-tolerant plants using HSP101 function? YES!! Overexpression of HSP101 increases thermotolerance in rice.

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