ABI4: A Tightly Controlled Regulator of Plant Stress Response - PowerPoint PPT Presentation

ABI4: A Tightly Controlled Regulator of Plant Stress Response Michelle Petitfils UC Santa Barbara Faculty Advisor: Ruth Finkelstein Dept: Molecular Cellular and Developmental Biology

Groundwater depletion

� Make plants more tolerant to drought and salinity � Need to understand pathways of response to stress hormones such as ABA � Can use that information to breed or construct stress-tolerant plants

Hormone in plants that inhibits germination and growth in unfavorable conditions. Plants become stress-adapted Plants rely on ABA so that they do not continue to grow and use up water when there is none The signaling pathways of ABA are not completely understood, but over 100 genes regulating ABA response are known

� ABI4 is both necessary and sufficient for ABA and other stress responses 3 uM ABA 4% Glucose Overexpress Wild-type Lack ABI4 abi4 Wildtype 6% Glucose

Although overexpression of GFP-ABI4 also confers ABA hypersensitivity, the GFP-ABI4 is Undetectable 35S ‐ ABI4 ‐ GFP GM Col 35S ‐ nuclearGFP

How is ABI4 regulated? Why is there less ABI4 than expected? Transcript Compare transcript vs. protein product to address pre- vs. post-translational control Use fusions to reporter gene (“GUS”) Protein

ABI4-GUS fusion is functional: overexpression confers hypersensitivity to ABA in root growth inhibition (and is detectable, but just barely) No transgene ABI4 fusion transgene ABA No ABA

� DNA RNA � ABI4-Gus Fusion Vs. Only Gus � If ABI4-Gus fusion transcript levels are much lower than those of GUS alone we know that ABI4 transcript accumulation is highly regulated

Determine Amount of Protein Determine Amount of Protein � RNA Protein � ABI4-Gus Fusion Vs. Only Gus � If GUS (activity) > GUS transcript ABI4-Gus protein ABI4-Gus transcript we know that ABI4-GUS protein accumulation is highly regulated (translation, activation, or stability)

� Beta-glucuronidase (GUS) � Detected both histochemically and fluorometrically, depending on substrate � Very stable reporter protein Best way found to determine change in protein concentration of ABI4 constructs

RESULTS: ABI4-GUS is highly regulated post-transcriptionally Background: Col (Wildtype) Transgene: ABI4pro-GUS ABI4pro-ABI4-GUS RNA ABI4-GUS transcript GUS transcript

RESULTS: ABI4-GUS is highly regulated post-transcriptionally rdr6 (Mutant: Allows us to detect ABI4) Background: Col (Wildtype) Transgene: ABI4pro-GUS ABI4pro-ABI4-GUS RNA (GUS transcript)

ABI4-GUS is highly regulated post-transcriptionally Background: Col rdr6 Transgene: ABI4pro-GUS ABI4pro-ABI4-GUS RNA (GUS transcript) Relative RNA: 1 6

ABI4-GUS is highly regulated post-transcriptionally Col rdr6 Background: Transgene: ABI4pro-GUS ABI4pro-ABI4-GUS RNA (GUS transcript) Relative RNA: 1 6 Protein 2175 0 97 (GUS activity)

ABI4-GUS is highly regulated post-transcriptionally Col rdr6 Background: Transgene: ABI4pro-GUS ABI4pro-ABI4-GUS RNA (GUS transcript) Relative RNA: 1 6 Protein 2175 0 97 (GUS activity) GUS Activity 2175 16 Transcript

Cycloheximide (chemical that inhibits protein synthesis) Stable: No Protein Degradation Cycloheximide RNA Protein Unstable: Protein Degraded

MG132 (Chemical that inhibits targeted protein degradation) Unstable: Protein Degraded MG132 (targeted) (misfolded)

CHX 35S-GUS GM 2000 1500 1000 500 0 GUS Activity

ABI4-GUS undergoes targeted degradation GM CHM MG132 MG132 + CHM 5hr 5hr 5hr 5hr

Which Parts of ABI 4 are destabilizing? ** * * Q P AP2 N-term - C-term Δ PEST (still has weak PEST?) Δ PEST/AP2 (still has weak PEST?) PEST N Δ PEST N Δ PEST/AP2

ABI4-GUS Degradation in Cycloheximide Activity 5 hrs CHM Activity 5 hrs control GUS Q P ** * * AP2 - N-term C-term Δ PEST Δ PEST/AP2 PEST N Δ PEST N Δ PEST/AP2 100% (No Degradation)

� Found that ABI4 is highly regulated post-transcriptionally � Discovered that ABI4 as a whole is targeted for degradation by the plant � Know that ABI4 instability is caused by more than one part of the ABI4

� How is ABI4 being targeted for destruction? � Which parts are most critical for instability? � What factors interact with destabilizing parts to target the protein for destruction? � Where does the ABI4 reside in the plant? � Is it always in the nuclei?

UC LEADS UC LEADS Ofelia Aguirre Ofelia Aguirre Eric Pressly Eric Pressly Dr. Ruth Finkelstein Dr. Ruth Finkelstein

Full Length T=O 2Hr Gm 2Hr CHM 4Hr GM N-terminal T=0 4Hr CHM 6Hr GM 6Hr CHM

CHM Time Course Results 7/22/09 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 t=0 2 hr GM 2 hr CHX 4 hr GM 4 hr CHX 6 hr GM 6 hr CHX 35S-ABI4- 35S-ABI4- 35S-ABI4- 35S-ABI4- 35S-ABI4- 35S-ABI4- 35S-ABI4- GUS GUS GUS GUS GUS GUS GUS

CHX+MG132 35S-ABI4-GR (background) MG132 CHX GM CHX+MG132 35S-ABI4-GUS MG132 CHX GM 250 200 150 100 50 0 GUS Activity