The Interactions of Plant Hormones and rop Genes By Malem Gutema Mentor Dr. John Fowler Dr. John Fowler Summer 2004

Rop Proteins Plants possess a special subfamily of GTPases denoted by Rop (Rho of Plants) proteins Plants possess a special subfamily of GTPases denoted by Rop (Rho of Plants) proteins Rop proteins have been first identified in pea (Yang and Watson), where they contribute to control tip growth in pollen tubes Rop proteins have been first identified in pea (Yang and Watson), where they contribute to control tip growth in pollen tubes G proteins or GTPases play a crucial role in the control of growth and shape of eukaryotic cells G proteins or GTPases play a crucial role in the control of growth and shape of eukaryotic cells GTPase protein

Signal Receptor ROP GDP Active GTP inactive - Control of growth and development - Stress responses - Disease resistance How does all it work? ROP

The plant hormone Abscisic Acid (ABA) ABA’s main roles - Regulating transpiration - Stress response Slow down growth - Slow down growth - - Germination of seeds.

The Plant hormone Auxin Auxin’s promotes initiation of - lateral roots - new leaves Auxin regulates elongation of Auxin regulates elongation of - stem - stem - root cells

Part I - Purpose Rop proteins are thought to interact with plant hormone signals, particularly during root growth. Rop proteins are thought to interact with plant hormone signals, particularly during root growth. Abscisic acid and auxin restrain root growth. Abscisic acid and auxin restrain root growth.

Hypothesis and Prediction We know ABA,Auxin We Hypothesize ABA, Auxin Our prediction ABA,Auxin Root growth Rop a symbol to show inhibition EVEN SHORTER Root growth

Part I - Experiments The experiments are designed to measure the effects of The experiments are designed to measure the effects of - (ABA) - Abscisic acid (ABA) - Auxin - Auxin Samples are Samples are - Wild-types - Wild-types - mutated at rop genes

Maize rop mutants There are nine rop genes in maize There are nine rop genes in maize I used samples with mutation of the rop 2, rop 6 and rop 7 genes I used samples with mutation of the rop 2, rop 6 and rop 7 genes How do you get the mutation? How do you get the mutation?

rop mutation Maps Exon Intron Transposable element

Root growth assay Root growth assay Question? Question? Would additional ABA and Auxin affect root growth? Would additional ABA and Auxin affect root growth? How are the wild-type and mutated samples affected? How are the wild-type and mutated samples affected?

Experiment set up Experiment set up Germinate seedlings in a water soaked paper towel Germinate seedlings in a water soaked paper towel Measure root length Measure root length before treatment Add auxin or ABA Add auxin or ABA Measure after 24 and 48hrs Measure after 24 and 48hrs Average the length Average the length

Auxin – rop 6/7 Mutant Effect !! rop 6/7 Double mutant SLOWER root growth 00.1 µM1.0 µM Auxin

Auxin rop 2 No Mutant Effect !!! rop 2 mutant SAME as wild-type µM 1.0 µM Auxin

ABA rop 2 Mutant Effect !! rop 2 mutant SLOWER root growth µM 500µM ABA

ABA- rop 6/ µM 500 µM ABA No Mutant Effect !!! rop 6/7 mutant SAME as wild-type

Summary of Results ABA, Auxin ABA, Auxin Auxinrop 6/7Supports hypothesis Auxinrop 6/7Supports hypothesis Auxinrop 2No effect ABArop 2Supports hypothesis ABArop 6/7No effect ABArop 2Supports hypothesis ABArop 6/7No effect Root growth Rop

Working Model ABA Root Growth Auxin rop 6/7 rop 2

Part II Do same root assay on a segregating population of rop2-m1 mutants Do same root assay on a segregating population of rop2-m1 mutants Take samples from an ear that has both homozygote and heterozygote Take samples from an ear that has both homozygote and heterozygote genotypes genotypes Do genotyping Do genotyping

What does segregating mean? rop2-m1/+Χ rop2-m1 / rop2-m1 rop2-m1 / rop2-m1 a homozygote rop2-m1 / rop2-m1 a homozygote rop2-m1 / + a heterozygote rop2-m1 / + a heterozygote Population is 50:50 homozygotes:heterozygotes + is a symbol for wild-type

Procedure Collect leaf samples Collect leaf samples Extract DNA Extract DNA Do a PCR reaction Do a PCR reaction with rop 2 primers with rop 2 primers Analyze the PCR product on an agarose gel to check the fragment sizes Analyze the PCR product on an agarose gel to check the fragment sizes

Detecting rop 2 mutations rop 2 primers Transposable element primer Mutation bands Wild type band

Tested hypothesis how rop genes inhibit plant hormones Tested hypothesis how rop genes inhibit plant hormones -Some data support -Some data don’t A more conclusive result will be obtained from the segregating experiment that is A more conclusive result will be obtained from the segregating experiment that is Coming soon……….. Coming soon……….. Summary Conclusion ABA rop 2 Auxin rop 6/7 Root Growth

Acknowledgments Ernest and Pauline Jaworski Fund Ernest and Pauline Jaworski Fund Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. John Fowler Dr. John Fowler Dr. Kevin Ahern Dr. Kevin Ahern Everybody at the lab Everybody at the lab - Zuzana Vejlupkova - Kirstin Arthur - Rex Cole