Transcript Analysis Under Drought Stress in Tall Fescue

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Transcript Analysis Under Drought Stress in Tall
Fescue

• Aaron Hightower
• Southeastern Oklahoma State University
• Noble Summer Scholar 2007
• Saha Lab

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Outline
Introduction
Materials Methods
Results Discussion
Summary
Future Work
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Importance

• Tall fescue is a cool season perennial grass
• 14 million hectares in United States
• Cattle feed
• Conservation
• Lawn and turf
• Drought is the single-most influential constraint
  on crop growth.
• Significantly decreases crop
• Production
• Persistence

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Drought Tolerance Mechanism

• Drought tolerance is a quantitative trait
  controlled by many genes and environmental
  factors. Tolerance mechanism relate to
• Specific protein production
• Increased water retention
• What controls these responses is the genetic
  reaction to conditions which code for up- and
  down-regulation of genes in order to increase
  survival.
• Transcript analysis over a period of drought
  stress is one way to observe the differentiation
  of expressed phenotypes due to genetic
  adjustments.

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Objectives

• To identify differentially expressed transcripts
  due to drought stress.
• Compare phenotypic expression between already
  identified susceptible and tolerant genotypes.
• Physiological observations and comparison between
  genotypes.
• Identify the utility of cDNA-AFLP in Tall Fescue.

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Outline
Introduction
Materials Methods
Results Discussion
Summary
Future Work
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Plant Material

• The two Tall Fescue genotypes analyzed were
  348(tolerant) and 947(susceptible).
• These were subjected to drought for two weeks.
• June 3 June 16

Day 14
348
947
Day 1
947
348
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Sample Harvest

• Plant Material cont.
• Tissues from both plants harvested daily for two
  weeks (June 3- June 16).
• Two samples
• Actively growing new shoots for RNA extraction.
• Leaf samples for Relative Water Content and
  Osmolality measurements.

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Day 1
Day 7
Day 14
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Measurements

• Relative Water Content (RWC)
• Cut four pieces from leaf samples (0.5-1g)
• Weighing of samples
• Fresh sample
• Immediately after harvest
• Turgid sample
• Soaked overnight in water
• Dry sample
• Dried overnight in vacuum oven at 60oC, 25Hg
• (Fresh wt. Dry wt.)
• (Turgid wt.- Dry wt.)

x 100 RWC
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Osmolality

• Sample Storage Preparation
• Leaf sample, after RWC samples taken, re-watered
• 3 mL of water into ziploc with leaf samples
• Soaked overnight
• Poured out excess water
• Put into -80oC

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Osmolality Measurement
Sample discs

• Vapor Pressure Osmometer
• Sample Prep
• Thawed leaf samples and blotted to dryness
• Put into syringe
• Sap extracted with plunger
• 10uL needed for VPO
• Placed sample on disc already on detector
• Closed and run lever locked to run
• Reading in mmol

Run lever
Sample Chamber
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RNA Extraction

• The RNA samples were collected into 2mL
  microcentrifuge tubes.
• Two steel beads were placed in tubes as well
• Used for grinding tissue
• Samples kept frozen in -80oC and when handled
  used liquid nitrogen to keep frozen
• Tubes put into shaker to grind up tissue
• RNA extracted using TRI Reagent (Molecular
  Research Center, Cincinnati, OH) according to
  manufacturers protocol.

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cDNA-AFLP Protocol

• Synthesize ss cDNA by Reverse Transcription
• Synthesize ds cDNA
• Quantify ds cDNA concentration
• Nano Drop 3000
• Restriction Digestion
• Using EcoRI and MseI enzymes
• Ligation of Adaptors
• Pre-amplification
• Less specifically amplified fragments
• Selective Amplification
• More specifically amplified fragments

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cDNA-AFLP Protocol cont.

• Performed cDNA AFLP on samples 348 and 947 for
  days 1, 7, and 14.
• Selective Amplification Eight different primer
  combinations used

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Primer Screening Genomic DNA

• Multiple primers were screened using the genomic
  DNA from both 348 and 947.
• Checked success by gel electrophoresis on 2
  Agarose with TAE buffer.
• For working primers, repeated with cDNA.
• Ran undiluted (1478.3ng/uL), 100ng/uL, and
  10ng/uL cDNA with working primer STS 303.
• The undiluted and 100ng/uL showed, 10ng/uL did
  not.

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Primer Screening cDNA

• Working primers
• Ran with cDNA (100-300ng/uL)
• Two reactions prepared, for 20 and 40 cycles for
  optimization.
• Verified by running on 2 Agarose with TAE
  buffer.
• 20 cycles faint, 40 cycles optimal.
• The primers which showed interesting
  characteristics were re-ran with primers to
  verify the results

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Outline
Introduction
Materials Methods
Results Discussion
Summary
Future Work
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Relative Water Content during two week water
stress

• Two genotypes did not differ significantly on
  RWC.
• Stable period, followed by sharp decline, then
  stabilized.
• The mechanism for drought tolerance in these
  genotypes is not a function of water content, but
  a different mechanism drives them.

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Humidity Temp.
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Primer Screening
348 947
348
947
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cDNA-AFLP Gel
348 D1 947 D1 348 D7 947 D7 348 D14 947 D14
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Polyacrylamide Score
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Outline
Introduction
Materials Methods
Results Discussion
Summary
Future Work
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Conclusion

• The Osmotic Potential further supports that
  previous studies have appropriately classified
  Tall Fescue 348 and 947 as drought tolerant and
  susceptible genotypes, respectively.
• There exists a correlation between humidity,
  temperature, and the Osmotic Potential of a
  plant.

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Conclusion cont.

• The observations have shown that drought narrowly
  affects the plants Relative Water Content during
  the first five days, but drastically affects it
  for subsequent days.
• The cDNA-AFLP has identified 73 up- and 8
  down-regulated fragments, which are good
  candidates for drought related gene studies.

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Outline
Introduction
Materials Methods
Results Discussion
Summary
Future Work
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Future Studies

• Further analysis of Osmotic Potential with regard
  to humidity and temperature and their effects
  upon Osmotic adjustment.
• Sequencing and identification of Differentially
  Expressed Transcripts.
• Verify the utility of the differentially
  expressed genes for drought screening.

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Acknowledgements Thanks

• Steve Rhines
• Emily Edwards
• Malay Saha
• Entire Saha Lab
• Francis Kirigwi
• Project supervisor
• Fellow Noble Scholars
• The Noble Foundation