Measuring Dynamic Biological Responses of Plants to Global Change using Short-lived Radioisotopes...

Measuring Dynamic Biological Measuring Dynamic Biological Responses of Plants to Global Responses of Plants to Global

Change using Short-lived Change using Short-lived RadioisotopesRadioisotopes

Calvin Howell

Duke University Physics

Triangle Universities Nuclear Laboratory

March 1, 2006 University of Notre Dame 2

OutlineOutline

• The TUNL-Phytotron Collaboration

• Motivation

• Status of Plant Studies with Radioisotopes

• Plant Physiology Basics

• Demonstration of Technique

• Immediate Plans


C.R. Howell (Physics)C. Reid (Biology)E. Bernhardt (Biology)A.S. Crowell (Physics Postdoc)M. Kiser (Physics graduate student)R. Phillips (Biology Postdoc)

TUNL-Phytotron Collaboration


What is a Phytotron?What is a Phytotron?

• CControlled EEnvironment FFacility• Growth chambers can control many factors:

– Soil type

– Air Temperature

– Light levels (total & UV)

– Carbon dioxide concentrationCarbon dioxide concentration

– Relative humidity

– Nutrients

– Air pollutants


OutlineOutline


• Motivation




• Immediate Plans


MotivationsMotivations

“Industrial Revolution”


MotivationsMotivations

Intergovernmental Panel on Climate Change (IPCC): Climate Change 2001, “The Carbon Cycle and Atmospheric Carbon Dioxide”

Climate models predict Climate models predict atmospheric COatmospheric CO22 levels levels

will double by the end will double by the end of this century!of this century!

How will plants respond?How will plants respond?


Carbon BudgetCarbon Budget

Sinks in units of billions of metric tons of carbon (GtC)Sinks in units of billions of metric tons of carbon (GtC)

Fluxes in units of billions of metric tons of carbon per year (GtC/year)Fluxes in units of billions of metric tons of carbon per year (GtC/year)



Interesting AsideInteresting Aside

• Total tonnage of CO2 produced by vehicles over 124,000 mile lifetime

• Assuming ~10 year lifetime, vehicles emit more than their own weight in CO2 per year

13 mpg

36 mpg

22 mpg

18 mpg

65 mpg

http://www.sierraclub.org/globalwarming/suvreport/pollution.asp


Grassland ResponseGrassland ResponseMultiple Factors:(C) CO2 ; 680 ppm(T) Temperature; +80 W/m2

(P) Precipitation; +50%(N) Nitrogen; +7g/m2 year

M. Rebecca Shaw et al., Science 298:1987-1990 (2002) – Carnegie Institute of Washington and Stanford Univ.


FACE StudiesFACE Studies• Free Air CO2 Enrichment (FACE)

experiments– Large-scale research programs to

study effects of increased CO2 levels– Many environmental variables– Difficult to correlate growth

parameters with high precision

• Findings from forest stands– Initially, carbon stored in woodInitially, carbon stored in wood– 2 years later, less found in wood, but 2 years later, less found in wood, but

more than double in fine rootsmore than double in fine roots– Nearly half of carbon uptake in short-Nearly half of carbon uptake in short-

lived tissues, such as foliagelived tissues, such as foliage– Increase in net primary production of Increase in net primary production of

25%25%– Growth rate increased about 26%Growth rate increased about 26%– Limited N Limited N no appreciable change no appreciable change

Duke FACTS-I Aerial ViewDuke FACTS-I Aerial View


FACE SitesFACE Sites


OutlineOutline


• Motivation




• Immediate Plans


Introduction to Plant Studies with Introduction to Plant Studies with RadioisotopesRadioisotopes

• 14C used in mid-1940’s – Long half-life (~5730 years)– Weak beta emitter– Tracer measured by destructive harvesting

• Use of 11C for in vivo studies demonstrated in 1963• 1973 – More and Troughton at the Department of

Scientific and Industrial Research in New Zealand showed that useful amounts of 11C can be produced using small van de Graaf accelerators– Labs in USA, Canada, Scotland, New Zealand, and Germany

start using 11C for mechanistic studies of photosynthate transport in the mid 1970’s

– Present studies at: Julich, Germany; Univ. Tokyo; BNL; TUNL-Duke


Features of using short-lived of using short-lived radioisotope tracersradioisotope tracers

• AdvantagesAdvantages– In vivo In vivo measurementmeasurement

– Use same specimen for Use same specimen for numerous experimentsnumerous experiments

– Conducive to studies Conducive to studies of dynamic phenomenaof dynamic phenomena

– Much greater Much greater sensitivity than that of sensitivity than that of carbon-14carbon-14

• ConsiderationsConsiderations– Experiments must be Experiments must be

performed near performed near acceleratoraccelerator

– Only observe short-Only observe short-term phenomena term phenomena

– For imaging, For imaging, sophisticated data sophisticated data acquisition and data acquisition and data analysis requiredanalysis required


Planned Research at the TUNL-Phytotron Facility

1. Studies of CO2 uptake and carbon translation under different environmental conditions

2. Root exudate measurements 3. Studies of exchange between plant roots and mycorhhiza

associations; ectomycorrhizal fungi (EMF)4. Nutrient uptake and translocation under different environmental

conditions


OutlineOutline


• Motivation




• Immediate Plans


Plant Physiology 101Plant Physiology 101

• Carbohydrates produced by photosynthesis

• Sugars produced in mature leaves and transported via phloem tissue

Light

H2O

CO2

Sugars

Chloroplasts trap light energy

6H6H22O + 6COO + 6CO22 + light + light C C66HH1212OO66 + 6O + 6O22

SugarSugar

From Discover Science, Scott, Foresman, & Co., 1993


Plant Physiology 101Plant Physiology 101

a) Sugars loaded into a sieve tubeb) Loading of the phloem sets up

water potential gradient that facilitates movement of water into dense phloem sap from the neighboring xylem

c) As hydrostatic pressure in phloem sieve tube increases, pressure flow begins, and sap moves through the phloem

d) At the sink, incoming sugars actively transported out of phloem and removed as complex carbohydrates

e) Loss of solute produces high water potential in phloem, and water passes out, returning eventually to xylem

http://home.earthlink.net/~dayvdanls/plant_transport.html


Phloem Transport BasicsPhloem Transport Basics

Sugars from LeafSugars from Leaf

StorageStorage

• Stems

• Roots

GrowthGrowth

• New Shoots

• Roots

ReproductionReproduction

• Seeds


OutlineOutline


• Motivation




• Immediate Plans


Carbon-11 ProductionCarbon-11 Production

p + 14N 11C + ++1

5

2 3 4

2

3

1 Produce H- ions in negative ion source

4

5

Accelerate H- ions toward +5MV terminal

Strip off electrons with carbon foil (H- p)

Accelerate protons away from +5MV terminal

Bend p in magnet and collide on 14N target


Production Block DiagramProduction Block Diagram

(CuO granules)

Average proton beam current = 1 ATotal irradiation time = 20 minutesGas cell pressure = 100 PSIGDesired activity = ~10 mCi

14N(p,)11C

T > 600ºC


1111C ProductionC Production

110 min10514 F

N

1

2/1

min034.02ln

11

tC 114

1411

tN

NC

]1[)0()()( 11

1414111111

t

NNCCCCeNtNtA

NN

CC

N

N

1414

1111


1111C PositronsC Positrons

+

1111C C 1111B + B + ++ + + ee

2115

116 ])()([ cmBmCmQ eNN

6

1

22112116 6)()(

iieN BcmcCmcCm

5

1

22112115 5)()(

iieN BcmcBmcBm

21111 ]2)()([ cmBmCmQ e

MeVQ 96.0


Development ExperimentsDevelopment Experiments

• Study barley plants grown in ambient (350 PPM) and elevated (700 PPM) levels of CO2

• Label plants under both conditions

• Analyze differences in carbon uptake and translocation


Single Detector MeasurementsSingle Detector Measurements

• Use detectors collimated for specific areas of plant to trace carbon allocation on a coarse (source/sink) scale

• Develop quantitative flow models to describe dynamics


Single Detector MeasurementsSingle Detector Measurements


Circuit DiagramCircuit Diagram

BGO DetectorBGO Detector

HVHV+1300V+1300V

Spect. Amp.Spect. Amp.

SCA Scaler


Qualitative ResultsQualitative Results

Data corrected for half-life and relative detector efficiency

BarleyGrown@350PPMLabeled@350PPM

BarleyGrown@700PPMLabeled@700PPM


Flow ModelFlow Model

Discrete observation times: tk where k = 0,1,2,.…Yk = counts in Sink B at time tk

Uk = counts in Total Sink at time tk

Leaf

Shoot

Root

Source

TotalSink

Sink A

Sink BSink B

Yk = - a1 Yk-1 - a2 Yk-2 - … - an Yk-n + b0 Uk + b1 Uk-1 + … + bm Uk-m

Input-Output AnalysisInput-Output Analysis:: (1)(1) Statistical, data-based modelingStatistical, data-based modeling(2)(2) No assumptions about mechanism(s) involvedNo assumptions about mechanism(s) involved


Flow ModelFlow Model

Best Model: YYkk = = -a-a22 Y Yk-2k-2 + b+ b00 U Ukk + b + b22 U Uk-2k-2

Extract Physically Significant Quantities:

(1)(1) GainGain – fraction of inputinput that shows up at the outputoutput(2)(2) Average transit timeAverage transit time


Flow ModelFlow ModelLeaf

Shoot

Root

Source

TotalSink

Sink A

Sink BSink B

Shoot Export

Treat entire plant asTreat entire plant as Total Sink Total Sink to probe leaf exportto probe leaf export

Leaf

Shoot

Root

Sink A’

TotalSink

Sink B’Sink B’Leaf Export


Modeling ProcedureModeling Procedure• Fit data with model using method of least squares

• This gives the model parameters a2, b0, and b2 and the statistical error in these parameters

• To determine the gain and average transit time, look at the output of the system with a unit impulse input

kU1 for k=0

0 for k022022 kkkk UbUbYaY

N

kkYGGain

0 G

YktTimeTransAvg

k

N

k 0..


One ExampleOne Example

5.1866

(0.0006)

0.6435

(0.0006)

0.43223

(0.00009)

-0.23666

(0.00008)

-0.69610

(0.00008)Run 2

7.7238

(0.0007)

0.6294

(0.0006)

0.29420

(0.00007)

-0.15852

(0.00006)

-0.78443

(0.00015)Run 1

< t > (min)Gb2b0a2

.)(0004.0.)(00997.063645.0 statsystG


ResultsResultsBest Model: YYkk = = -a-a22 Y Yk-2k-2 + b+ b00 U Ukk + b + b22 U Uk-2k-2

[CO2] (ppm)

Age (days)

Leaf Export Fraction

Shoot Export Fraction

Leaf-to-Shoot Transit

Time (min)

Shoot-to-Root Transit Time (min)

350350 10-1210-12 0.78 ± 0.030.78 ± 0.03 0.28 ± 0.010.28 ± 0.01 20.39 ± 5.0220.39 ± 5.02 6.78 ± 2.306.78 ± 2.30

700700 10-1210-12 0.90 ± 0.030.90 ± 0.03 0.64 ± 0.010.64 ± 0.01 17.71 ± 1.0317.71 ± 1.03 6.45 ± 1.276.45 ± 1.27

350350 18-2118-21 0.92 ± 0.050.92 ± 0.05 0.39 ± 0.030.39 ± 0.03 25.03 ± 1.3425.03 ± 1.34 6.48 ± 0.016.48 ± 0.01

700700 18-2118-21 0.80 ± 0.030.80 ± 0.03 0.52 ± 0.0050.52 ± 0.005 22.01 ± 8.4122.01 ± 8.41 15.76 ± 2.9915.76 ± 2.99


2D Imaging2D Imaging

● Approximate plant as planar source● Build up image through a sequence of exposures● Enhanced spatial resolution via coincidence detection

CsF detectors-High stopping power-High count rate capability


Then We Have…Then We Have…

Leaf

Shoot

Root

Source

TotalSink

More Accurate

Flow Model

Enhanced ResolutionEnhanced Resolution Observe Fine Details of Observe Fine Details of Dynamic BehaviorDynamic Behavior


2D Imaging2D Imaging


Coincidence CircuitCoincidence Circuit


EfficiencyEfficiency

• Some pixels “see” more of the array than others• Account for this by simulations



From Above

From Side


Prototype EfficiencyPrototype Efficiency

x (cm)

y (c

m) x (cm)

y (cm)

W

W


Spatial Probability DistributionsSpatial Probability Distributions

11 22

33 44 55

66 77 88

99 1010

1111 1212

1313 1414

1515 1616 1717

1818 1919 2020

2121 2222

2323 2424


Prototype ResolutionPrototype Resolution

W

W

x (cm)

y (cm)

x (cm)

y (c

m)


Image ReconstructionImage Reconstruction

(1)(1) Add SPD for each Add SPD for each coincidence event for a coincidence event for a given exposure timegiven exposure time

(2) Subtract off background events scaled to the exposure time

(3) Correct for relative detection efficiency

(4) Correct for 11C half-life each minute of exposure

x (cm)

y (c

m)



(1) Add SPD for each coincidence event for a given exposure time

(2)(2) Subtract off background Subtract off background events scaled to the events scaled to the exposure timeexposure time



x (cm)

y (c

m)





(3)(3) Correct for relative Correct for relative detection efficiencydetection efficiency


x (cm)

y (c

m)






(4)(4) Correct for Correct for 1111C half-life C half-life each minute of exposureeach minute of exposure

x (cm)

y (c

m)


For ExampleFor Example

x (cm)

y (c

m)


Immediate PlansImmediate Plans

• Install radioactive handling system

• Develop root exudate experiment

• Build high-resolution 2D PET imager

• Start full research program


Radioisotope Production

1. 11CO2 (half life = 20 min.)

14N + p 11C + Target: gas

3. 18F- (half life = 109 min.) 18O + p 18F + n

Target: 18O enriched water

2. 13NO3- (half live = 10 min.)

16O + p 13N + Target: 18O depleted water

4. H218O (half life = 2 min.)

16O + p 15O + d

Target: water

March 1, 2006 University of Notre Dame 57Ep (MeV)

14N(p,)11C Cross Section


Radioactive Materials Handling System


Root Exudate Experiment


High resolution 2D imagers

5 cm x 5 cm x 1.5 cm2mm x 2mm pixels (0.1 mm gap)

20 cm x 30 cm field of view

Measuring Dynamic Biological Responses of Plants to Global Change using Short-lived Radioisotopes...

Documents

Transcript of Measuring Dynamic Biological Responses of Plants to Global Change using Short-lived Radioisotopes...