LOCATION OF GTL-1 GENE EXPRESSION IN PLANTS & DROUGHT STRESS REGULATION OF GTL-1 GENE EXPRESSION

Gretchen Booher Kelci Adam

Ben Hoffherr Chester Trocha

GTL-1 BACKGROUND INFORMATION

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.

GTL - 1 BACKGROUND INFORMATION

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

0 1 3 6 12

Dehydration (hr)

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

Col-0 gtl1-4

GTL-1 mutants show lower transpiration possibly through lower number of stomata in leaves

HYPOTHESIS

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.

MATERIALS

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

METHODS

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?

METHODS 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

RESULTS 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

RESULTS 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

Top Bottom Petole0

0.5

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1.5

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2.5

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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.

RESULTS FOR PART 1– LETS TAKE A LOOK! Is the GTL-1 gene expressed more in bottom

epidermal layer than top epidermal layer? Yes!

Top Leaf- Normal Watering Bottom Leaf- Normal Watering

RESULTS FOR PART 1– LETS TAKE A CLOSER LOOK! 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

RESULTS FOR PART 2

Normal Stress0

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Series1

Blu

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nte

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y R

ati

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Does Drought treatment reduce GUS staining ? YES!!! Overall blue staining has decreased, therefore

decreasing the overall expression of GTL-1.

Bottom Leaf – Normal watering Bottom Leaf - Drought Stressed

Overall staining has decreased even in bottom epidermal layers

RESULTS FOR PART 2 – LET’S TAKE A LOOK!

2.9kb GTL1 promoter is functional promoter in terms of drought response for GTL1 expression

IN CONCLUSION…

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 expressionThis data implicates that GTL-1 regulates stomatal development in response to drought treatment.

FURTHERING RESEARCH

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