The Role of Cytokinin In the Growth-Defense Tradeoff

The Role of Cytokinin In the Growth-Defense TradeoffKatie ClearyLab Meeting 4-21-16

Presentation overviewPlant defense backgroundInformation pertinent to experimentWalk-through of experiment steps, results, analysisMass/area assays and evaluationFuture plans

Arabidopsis mutants displaying constitutive activation of defense responses show reduced growth

High SA mutants

DefenseGrowth

Cytokinin is important for plant growth and yield

Yield QTL mapped to a gene encoding a cytokinin degradation enzyme, OsCKX2lonely guy (log) rice mutant is unable to produce active cytokininsAshikari et al., 2005Kurakawa et al., 2007

Important information:Col-0: wild type Arabidopsis ahk2,3: cytokinin receptor mutantFlg22: 22 amino acid peptide from the protein that constitutes bacterial flagellumcytokinin: plant hormone involved in defense and growth

Preliminary ExperimentCol-0 and ahk2,3Spray: control groups: H2Oexperimental groups: 100M flg22Both treatments sprayed only once to plantsPictures taken each M,W,F

Results from Preliminary ExperimentCol-0ahk2,3H2O

flg22H2O

flg22

Preliminary ExperimentProblems with the preliminary experimentSurface tension of flg22 water suspension led to uneven flg22 application >> Decided to add SilwetDose: Decided to change 100M >>100nMDose frequencyDecided to spray every M,W,F and try continuous growth on agar with flg22

(Gomez-Gomez et al. 1999)

Experiments 1 & 2Experiment 1Experiment 2Plants grown on soilSprayed with 100nM flg22 every Monday, Wednesday, FridayPictures taken once a week1-4 control: H2O + Silwet5-9 treatment: flg22 + SilwetPlants grown on MS agarPlants grown on MS media containing 100nM flg22 (constant)Pictures taken once at the end of experiment

Experiment 1Col-0ahk2,3H2O

flg22H2O

flg22

Experiment 1

Experiment 1Col-0 had a % growth reduction more than 10 times higher than ahk2,3 This suggests cytokinin mediates the growth-defense tradeoffahk2,3 was not inhibited because it cannot perceive cytokininCol-0 was inhibited because it can perceive cytokinin

Experiments 1 & 2Experiment 1Experiment 2Plants grown on soilSprayed with 100nM flg22 every Monday, Wednesday, FridayPictures taken once per week1-4 control: H2O + Silwet5-9 treatment: flg22 + SilwetPlants grown on MS agarPlants grown on 100nM flg22 in MS media (constant)Pictures taken once at the end of experiment (22 days)

Experiment 2Col-0ahk2,3

100nM100nM0nM0nM

Experiment 2** second ahk2,3 column represents % reduction calculated with plate #2 thrown out due to contamination

Experiment 222 days2nd ahk2,3 plate is an outlierSuggests that cytokinin mediates the growth defense tradeoffAdjusted ahk2,3 % reduction shows plants treated with flg22 were actually larger than those untreated

Experiment 3Plants grown on MS agarPlants grown on MS media containing 100nM flg22 (constant)Plants grown on MS media containing 500nM flg22 (constant)Pictures taken once at the end of experiment (16 days)

Experiment 3Col-0ahk2,3

100nM100nM500nM500nM0nM0nM

Experiment 3

= p < 0.05

Experiment 316 daysCytokinin likely mediates growth inhibition during defense Col-0 % reduction in 500nm flg22 was double the growth reduction seen in Col-0 treated with 100nM flg22ahk2,3 plants treated with 500nM flg22 were actually larger than ahk2,3 treated with100nM flg22

Mass AssaysFlg22cytokinin

Col-0 treated with water vs. flg22

Col-0 and ahk2,3 treated with cytokinin

Evaluation of assaysArea analysis with ImageJ requires tracing the outline of the plantMore human errorMass measured using same scaleLess human errorBetter methodWhat needs to happen now?Mass assay: Col-0 and ahk2,3 treated with flg22

Future of this experimentRepeat area assaysMore platesFewer plants per plate16 days (as in Experiment 3)Repeat mass assays on larger, more complete scale

What Im doing next

Thank you