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

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Jan 16, 2024 373 likes •697 views

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

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Michelle Petitfils UC Santa Barbara Faculty Advisor: Ruth Finkelstein Dept: Molecular Cellular and Developmental Biology

ABI4: A Tightly Controlled Regulator of Plant Stress Response

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Groundwater depletion

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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

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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

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ABI4 is both necessary and sufficient for ABA

and other stress responses

Wild-type Lack abi4 3 uM ABA

Wildtype 4% Glucose

6% Glucose Overexpress ABI4

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Col 35S‐nuclearGFP

35S‐ABI4‐GFP GM

Although overexpression of GFP-ABI4 also confers ABA hypersensitivity, the GFP-ABI4 is Undetectable

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How is ABI4 regulated? Why is there less ABI4 than expected?

Compare transcript

vs. protein product to

address pre- vs. post-translational control Use fusions to reporter gene (“GUS”)

Transcript Protein

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No transgene ABI4 fusion transgene

ABI4-GUS fusion is functional:

verexpression confers hypersensitivity to ABA in root

growth inhibition (and is detectable, but just barely)

No ABA ABA

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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

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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)

Determine Amount of Protein Determine Amount of Protein

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Beta-glucuronidase (GUS) Detected both histochemically and

fluorometrically, depending on substrate

Very stable reporter protein

Best way found to determine change in protein concentration

f ABI4 constructs

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RNA

Col (Wildtype)

ABI4pro-GUS ABI4pro-ABI4-GUS

Background: Transgene:

RESULTS: ABI4-GUS is highly regulated post-transcriptionally

GUS transcript ABI4-GUS transcript

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RNA (GUS transcript)

Col (Wildtype) rdr6 (Mutant: Allows us to detect ABI4)

ABI4pro-GUS ABI4pro-ABI4-GUS

Background: Transgene:

RESULTS: ABI4-GUS is highly regulated post-transcriptionally

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RNA (GUS transcript)

Col rdr6

ABI4pro-GUS ABI4pro-ABI4-GUS

Background: Transgene:

Relative RNA: 1 6

ABI4-GUS is highly regulated post-transcriptionally

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RNA (GUS transcript) Protein (GUS activity) Col rdr6 ABI4pro-GUS ABI4pro-ABI4-GUS

Background: Transgene:

2175 0 97 Relative RNA: 1 6

ABI4-GUS is highly regulated post-transcriptionally

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

ABI4-GUS is highly regulated post-transcriptionally

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RNA Cycloheximide (chemical that inhibits protein synthesis)

Cycloheximide

Protein Unstable: Protein Degraded Stable: No Protein Degradation

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(targeted) (misfolded) MG132

MG132 (Chemical that inhibits targeted protein degradation)

Unstable: Protein Degraded

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

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ABI4-GUS undergoes targeted degradation

GM CHM MG132 MG132 + CHM 5hr 5hr 5hr 5hr

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P AP2

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N-term C-term

PEST ΔPEST/AP2 (still has weak PEST?) ΔPEST (still has weak PEST?)

N ΔPEST N ΔPEST/AP2

Which Parts of ABI 4 are destabilizing?

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P AP2

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N-term C-term

PEST ΔPEST/AP2 ΔPEST

N ΔPEST N ΔPEST/AP2

GUS

ABI4-GUS Degradation in Cycloheximide

100% (No Degradation)

Activity 5 hrs CHM Activity 5 hrs control

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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

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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?

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UC LEADS UC LEADS Ofelia Aguirre Ofelia Aguirre Eric Pressly Eric Pressly

Dr. Ruth Finkelstein
Dr. Ruth Finkelstein

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

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CHM Time Course Results 7/22/09

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 t=0 2 hr GM 2 hr CHX 4 hr GM 4 hr CHX 6 hr GM 6 hr CHX 35S-ABI4- GUS 35S-ABI4- GUS 35S-ABI4- GUS 35S-ABI4- GUS 35S-ABI4- GUS 35S-ABI4- GUS 35S-ABI4- GUS

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50 100 150 200 250 GM CHX MG132 CHX+MG132 GM CHX MG132 CHX+MG132 35S-ABI4-GUS 35S-ABI4-GR

GUS Activity

(background)