THE EFFECT OF ATR ON UV-INDUCED STEM CELL DEATH IN ARABIDOPSIS

Robert UrsuDr. John Hays

Oregon State University

IN MY LAB…

I work with Arabidopsis thaliana

Entire genome is sequenced, thus

Great model organism to study

RELEVANCE Better grasp of UV damage My research translates into human cell

signaling and would be important in contributing to ageing research development

Environment can benefit greatly from increased amount of knowledge of plants

Understanding of plants can help boost crops, thus economy

WE STUDY SIDE EFFECTS OF ARABIDOPSIS MUTATIONS

I obtain Arabidopsis seeds with a “T-DNA” (tumefacien DNA) insertion

I raise them to maturation to obtain more seeds and/or do any crossing with plants that have other T-DNA insertions

I observe the amount of stem cell death at the root tip after UV-radiation

WHY THE STEM CELL ROOT TIP AND WHY UV RADIATION?

Vascular

Cortical / Endodermal

Columellar / Central Root Cap

Epidermal / Lateral Root Cap

Root tip is the site where replication is most active

UV irradiation mimics the damage done from common, yet harmful sun rays

Damage done during replication is what we are most interested in

WE STUDY THEM WITH

Microscopy – dye the root andobserve under a microscope.

PCR (Polymerase chain reaction) – amplifies DNA to perform a “gel electrophoresis”.

Gel electrophoresis – tells me if the plant in question has the T-DNA insertion

PCR Mechanics

Atr Gene

WT Primer 1

WT Primer 2

Product = WT allele

Atr Gene

T-DNA Primer 1

T-DNA Primer 2

T-DNA Insertion

Product = T-DNA allele

PCR & GEL ELECTROPHORESIS EXAMPLE

WT control tDNA control

WT negative

LaddertDNA positive

WT positive tDNA positive

WT positive tDNA negative

Plant 1

Plant 2

Etc.

MY SPECIFIC PROJECT INVOLVES… SOG-1 transcription factor

- Mainly responsible for initiating PCD (programmed cell death)

Kinase ATR (type of enzyme) - Helps locate and repair damage on replicating single strand DNA

ATR can signal SOG-1 to carry out PCD

SOG1: EXCEPTION TO GENOTYPING

Is a single base mutation instead of a strand of DNA along the allele

Must sequence the PCR DNA product with sequencing machine to locate base mutation

MY SPECIFIC PROJECT Kinase ATR

ATR is Not Activated

ATR is Activated

Programmed Cell Death Damage is Repaired

ATR directly phosphorylates helicase to stall DNA “unwinding”

MY SPECIFIC PROJECT

I am comparing the stem cell death of plants that are Wt (control) to plants that are ATR-/-SOG1-/- using microscopy

HYPOTHESIS

Significant UV induced stem cell death within an Arabidopsis plant that is ATR-deficient is a response dependent upon the SOG1 transcription factor

PREDICTION The plants that are ATR-/-SOG1-/- deficient

will experience no significant cell death following UV radiation compared to the wt plants.

Col-0 Atr SOG-1 Atr-SOG-10

1

2

3

4

5

6

PCD 24 Hours Post UV Irradiation

Plant Deficiency

Avera

ge S

tem

Cell

Death

EXPERIMENT

After Wt and ATR-/-SOG1-/- plants grew for a few days, I UV irradiate them using consistent amount of energy.

Counted stem cell death in root tip of plants using microscopy.

RESULTS

Col-0 Atr SOG-1 Atr-SOG-10

1

2

3

4

5

6

PCD 24 Hours Post UV Irradiation

Plant Deficiency

Ave

rag

e S

tem

Ce

ll D

ea

th

ANALYZATION

Col-0 Atr SOG-1 Atr-SOG-10

1

2

3

4

5

6

PCD 24 Hours Post UV Irradiation

PredictedActual

Plant Deficiency

Avera

ge S

tem

Cell D

eath

EXPLANATION

Plant that I thought was Atr-/- and SOG1 -/- was actually SOG-1 +/-. So only ¼ of its offspring was SOG-1 -/-

Plant was Atr-/- and SOG1 -/- and stem cell death of an Atr deficient plant is not dependant on SOG-1 transcription factor

OR

ACKNOWLEDGMENTS Colin Tominey Peter Hoffman Buck Wilcox Marc Curtis Dr. John Hays Kevin Ahern HHMI