T HE E FFECT OF ATR ON UV- I NDUCED S TEM C ELL D EATH IN A RABIDOPSIS Robert Ursu Dr. John Hays Oregon State University

I N MY LAB … I work with Arabidopsis thaliana Entire genome is sequenced, thus Great model organism to study

R ELEVANCE 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

W E STUDY SIDE EFFECTS OF A RABIDOPSIS 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

W HY 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

W E STUDY THEM WITH Microscopy – dye the root and observe 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 & G EL E LECTROPHORESIS E XAMPLE WT controltDNA control WT negative Ladder tDNA positive WT positive tDNA positive WT positive tDNA negative Plant 1 Plant 2 Etc.

M Y SPECIFIC PROJECT I NVOLVES … 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

S OG 1: E XCEPTION 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

M Y 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”

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

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

P REDICTION The plants that are ATR-/-SOG1-/- deficient will experience no significant cell death following UV radiation compared to the wt plants.

E XPERIMENT 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.

R ESULTS

A NALYZATION

E XPLANATION 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

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