1. Plant Thermotolerance - hot1-3 (HSP101) HORT 301 9/30/2008

2. Your goal: Discover New Genes that function for Thermotolerance Choose one of the following: Reverse genetic approach Forward genetic approach

3. Reverse genetic approach for thermotolerance mutant 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.

4. 2.Generate mutants or overexpression lines 3.Test hypothesized responses (Thermotolerance) Control Over- expression Co- suppression Mishra et al., 2002 Genes and Development Back

5. Forward genetic approach for thermotolerance mutant 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 24  C45  C 2 hr38  C 1.5hr 45  C 2 hr Chan Yul’s experiment

6. 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)

7. 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

8. 4. Map and clone the mutation Jander et al., 2002 Plant Physiol

9. 5. Confirm by Complementation test Hong and Vierling, 2000 PNAS So, HSP101 gene functions for acquired thermotolerance

10. 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

11. HSP101 Function of HSP101 : Molecular chaperone; ATP-dependent dissociation of protein aggregates Baniwal et al., 2004 J. Biosci.

12. 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. Back