1. L OCATION OF GTL-1 GENE EXPRESSION IN PLANTS & DROUGHT STRESS REGULATION OF GTL-1 GENE EXPRESSION Gretchen BooherKelci Adam Ben Hoffherr Chester Trocha

2. GTL-1 B ACKGROUND I NFORMATION The development of crops that have a higher water use efficiency is a high priority world wide. However, the genetic basis for how plants regulate water usage is not known. GTL1 has been identified as a gene that regulates water use and drought tolerance. More specifically, GTL-1 regulates stomatal number and transpiration. The reporter gene GUS is used to highlight the location of the GTL1 expression. Source: Chan Yul Yoo- Center for Plant Environmental Stress Physiology, Purdue University.

3. GTL - 1 B ACKGROUND I NFORMATION Source: Chan Yul Yoo- Center for Plant Environmental Stress Physiology, Purdue University. GTL-1 mutations (gtl1-4 and gtl1-5) inhibit GTL1 expression GTL1 expression is negatively regulates by dehydration treatment GTL1 COR15a ACT2 013612 Dehydration (hr)

4. GTL-1 mutants improve drought tolerance by maintaining leaf relative water content during drought

5. Col-0gtl1-4 GTL-1 mutants show lower transpiration possibly through lower number of stomata in leaves

6. H YPOTHESIS The GTL1 Gene will be expressed in the leaf epidermal tissues to regulate stomatal development. Drought stress in soil is responsible for decreasing the expression of the GTL1 gene.

7. M ATERIALS Transgenic Arabidopsis harboring GUS gene driven by GTL1 promoter 70% ethanol 90% acetone GUS staining solution X-Gluc Oven set at 37º C Microscope Digital Camera

8. M ETHODS Part 1 – Normal Watering of Plants Watering transgenic plants normally 3 Plant tissue samples from each individual plant Peel for Top of Leaf Peel for Bottom of Leaf Cut section of Petiole Samples placed in Acetone Rinsed in Ethanol Placed in GUS Stain and X-Gluc overnight in 37ºC in the oven Rinsed in Ethanol Observed under microscope – What did we find?

9. M ETHODS Part 2 – Drought Stress Transgenic plants were not watered (drought stressed) 3 Plant tissue samples from each individual plant Peel for Top of Leaf Peel for Bottom of Leaf Cut section of Petiole Samples placed in Acetone Rinsed in Ethanol Placed in GUS Stain and X-Gluc overnight in 37ºC in the oven Rinsed in Ethanol Observed under microscope

10. R ESULTS ANALYSIS METHOD How do we measure the GUS expression? 1. The blue intensity scale  determine by eyes and quantify the intensity 1 2 3 2. Detailed observation using a microscope

11. R ESULTS FOR PART 1 Where is GTL-1 Gene expressed in the Plant??? LEAVES, NOT in the Petiole!!! On both the Top and Bottom, but bottom layers showed a little stronger expression However, there is a problem! Because GUS expression is very strong in mesophyll layers, it’s hard to determine what is exactly stained. So we observed epidermal layers using a microscope to determine the exact staining location.

12. R ESULTS FOR PART 1– L ETS T AKE A L OOK ! Is the GTL-1 gene expressed more in bottom epidermal layer than top epidermal layer? Yes! Top Leaf- Normal WateringBottom Leaf- Normal Watering

13. R ESULTS FOR PART 1– L ETS T AKE A C LOSER L OOK ! Is the GTL-1 gene expressed guard cells? Yes, it is expressed in guard cells and neighboring cells, but not in pavement cells. guard cells neighboring cells pavement cells

14. R ESULTS FOR PART 2 Does Drought treatment reduce GUS staining ? YES!!! Overall blue staining has decreased, therefore decreasing the overall expression of GTL-1.

15. Bottom Leaf – Normal wateringBottom Leaf - Drought Stressed Overall staining has decreased even in bottom epidermal layers R ESULTS FOR PART 2 – L ET ’ S TAKE A LOOK ! 2.9kb GTL1 promoter is functional promoter in terms of drought response for GTL1 expression

16. I N C ONCLUSION … Wild Type gtl1 Mutant Higher GTL1 expression in bottom epidermal layer No or lower GTL1 expression Higher Stomatal number Lower Stomatal number Higher Transpiration Lower Transpiration Lower drought tolerance Higher drought tolerance Which plant is more ideal??? 1.GTL-1 is expressed mainly in guard cells and neighboring cells in bottom epidermal layers of leaf 2.Drought treatment decreases GTL-1 expression This data implicates that GTL-1 regulates stomatal development in response to drought treatment.

17. F URTHERING R ESEARCH What other environmental factors could regulate GTL-1 expression? Salinity Highly organic soils Temperature What is a more scientific way of measuring blue stain? Measure stained area Count stained stomatal number / area Inclusion of the measured transpiration rate. Could give more concrete evidence