Using fast repetition rate fluorometry to estimate PSII electron flux per unit volume
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Transcript of Using fast repetition rate fluorometry to estimate PSII electron flux per unit volume
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Using Fast Repetition Rate
fluorometry to estimate PSII electron
flux per unit volume: A purely optical method for estimating GPP?
Kevin Oxborough
Principal Scientist
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Kevin Oxborough (CTG)
Mark Moore (Southampton)
Dave Suggett, Richard Geider (Essex)
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H2O
PQ
e-
PC
NADP
e-
PSII PSIb6f
CO2 assimilation
e-
PC
PQH2
Photosynthetic electron transport
e-PSII flux
FRRfhn hn
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JVPII = 𝑎PII′ ∙ 𝐸
𝜎PII′ = absorption cross section of PSII photochemistry (m2 PSII-1)
𝑎PII′ = absorption coefficient for PSII photochemistry (m-1)
𝐸 = Photon irradiance (photons m-2 s-1)
FRRf FRRf
JPII = 𝜎PII′ ∙ 𝐸
From PSII electron flux to GPP
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H2O
PQe-
Open RCII
Photochemistry and fluorescence
p
f
d How consistent is this relationship?
ϕ𝑓 =𝑘𝑓
𝑘𝑝 + 𝑘𝑓 + 𝑘𝑑
ϕ𝑝 =𝑘𝑝
𝑘𝑝 + 𝑘𝑓 + 𝑘𝑑
𝑃
𝐹∝𝑘𝑝
𝑘𝑓
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Closed RCII
Photochemistry and fluorescence
f
d
H2O
PQ
ϕ𝑓 =𝑘𝑓
𝑘𝑓 + 𝑘𝑑
ϕ𝑝 = 0
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The FRRf technique
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Open RCIIs
Closed RCIIs
Saturation phase:200 µs sequence of
100 x 1 µs 'flashlets' on a 2 µs pitch
Absorption cross section of
PSII photochemistry
Fo or F'
Fm or Fm'
sPII or sPII'
Single turnover (ST) FRRf measurement
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Saturation phase100 x 1 µs flashlets
2 µs pitch
Relaxation phase40 x 1 µs flashlets
50 µs pitch
Saturation and relaxation phases
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100 x 1 µs flashlets
2 µs pitch
40 x 1 µs flashlets
50 µs pitch
Saturation and relaxation phases
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Physical cross section of the PSII
light-harvesting system (LHII)
Absorption cross section of
LHII (sLHII)
Absorption cross section of
PSII photochemistry (sPII)
All unit area (m2 PSII-1)
Absorption cross section (Sigma)
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The FRR-ST data fit
Kolber, Prášil and Falkowski (1998)
𝐶𝑛 = 𝐶𝑛−1 + 𝑅𝜎PII ∙1 − 𝐶𝑛−1
1 − 𝐶𝑛−1 ∙ 𝑝
Fn = Fo + Fm − Fo ∙ 𝐶𝑛 ∙1 − 𝑝
1 − 𝐶𝑛 ∙ 𝑝
𝐶𝑛 = closed RCII at flashlet 𝑛𝑅𝜎PII = RCII closed by first flashlet
𝑝 = RCII connectivity
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RsPII
• Probability of an RCII being closed by the first flashlet in an FRR-ST sequence
• Dimensionless parameter• Workable range of 0.03 to 0.06• Can be 'set' by changing ELED
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RsPII – optimum ELED intensity
Super-optimalRsPII = 0.0774
Fv /Fm = 0.569
Sub-optimalRsPII = 0.0258
Fv /Fm = 0.561
OptimalRsPII = 0.0496
Fv /Fm = 0.573
Increasing ELED
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Connectivity among RCIIs
Separate Package
Open RCII
Closed RCII
LHII
Lake
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Open RCII
Closed RCII
LHII
Partial connectivity
Dimer model
Connectivity among RCIIs
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Photosystem II dimer
taken from Nield & Barber (2006)
• X-ray crystallography• Core from cyanobacterium• LHCII from spinach
• Cryo-EM from spinach
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Basic terminology
ParameterPSII photochemistry
PSII light harvesting
Absorption cross section (m-2 PSII-1) sPII(′) sLHII
Absorption coefficient (m-1) 𝑎PII(′) 𝑎LHII
Photon efficiency (dimensionless) fPII(′)
σPII′ = σLHII ∙ ϕPII′
𝑎PII′ = 𝑎LHII ∙ ϕPII′
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Flux terminology
Flux PSII electron flux Photon flux
Defined area JPII (e- PSII-1 s-1)
Unit area E (photons m-2 s-1)
Unit volume JVPII (e- m-3 s-1)
JPII = 𝜎PII′ ∙ 𝐸
JVPII = 𝑎PII′ ∙ 𝐸
e- PSII-1 s-1 m2 PSII-1 · photons m-2 s-1
e- m-3 s-1 m-1 · photons m-2 s-1
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JVPII = 𝑎PII′ ∙ 𝐸
FRRf FRRf
JPII = 𝜎PII′ ∙ 𝐸
From PSII electron flux to GPP
= 𝑎LHII ∙ 𝜙PII′ ∙ 𝐸Sigma method
Absorption method
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JVPII = sPII' · [RCII] · (1 – C ) · E
sPII' from iterative curve fit to FRR-ST data
[RCII] from chlorophyll determination (nPSII)
(1 – C ) from light + dark FRR data
E from PAR sensor
Calculation of PSII electron flux per unit volume (JVPII) using the sigma method
Kolber, Prášil and Falkowski (1998)
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The sigma method (Kolber et al. 1998)
Can be applied on wide spatial and temporal scales
Can be used to probe PSII photochemistry
Provides a good estimate of PSII electron flux (JPII)
Estimate of rETR (relative electron transport rate to NADP)
• JVPII requires an independent estimate of [RCII]
• Requires an assumed level of connectivity to quantify C
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The absorption method (Oxborough et al. 2012)
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H2O
PQ
Open RCII
Basic principle of the absorption method
e-
p
f
d
dfp
f
fkkk
k
f
dfp
p
pkkk
k
f
𝑃
𝐹∝𝑘𝑝
𝑘𝑓
Absorption method requires that this is a consistent relationship
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Consequence:
RCII =𝐹𝑜𝜎PII
∙KR
𝐸LED
Calculation of [RCII] using the absorption method
Hypothesis:
𝑘𝑝
𝑘𝑓falls within a very narrow range
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NIOZ PROTOOL workshop, Yerseke (2012)
12 phytoplankton speciesChaetoceros sp.
Ditylum brightwellii
Emiliania huxleyi
Nannochloropsis gaditana
Phaeocystis glabosa
Prorocentrum sp.
Skeletonema sp.
Synechococcus (red 9903 + green 0417)
Tetraselmis sp.
Thalassiosira pseudonana (-Fe / +Fe)
Thalassiosira westfloggii (-Fe / +Fe)
MethodsFast Repetition Rate fluorometers
Mk I and Mk II FASTtracka
FastOcean
Flash O2 release
Thermoluminescence
MIMS13C
Absorption spectrometry
Fluorescence excitation spectrometry
Organised by: Greg Silsbe & Jacco Kromkamp
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[RCII] x 1016 m-3
F o/ s
PII
NIOZ PROTOOL workshop, Yerseke (2012)
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Implications for the sigma algorithm
JVPII = sPII' · [RCII] · (1 – C ) · E
sPII' from iterative curve fit to FRR-ST data
[RCII] from FRR-ST data
(1 – C ) from light + dark FRR data
E from PAR sensor
No longer a requirement for independent measurement of [RCII]
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The absorption algorithm
Consequence:
JVPII = oF′ ∙KR
ELED∙ 𝐸
Hypothesis:
𝑘𝑝
𝑘𝑓falls within a very narrow range
Where oF' is the emission from open RCII under ambient light
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Comparing the sigma and absorption algorithms
oF′ = Fo ∙𝜎PII′
𝜎PII∙ 1 − 𝐶
oF′ =Fm ∙ FoFm − Fo
∙Fm′ − F′
Fm′
(Sigma algorithm)
(Absorption algorithm)
Absorption algorithm doesn't require sPII, sPII' or 1 – C
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Comparing the sigma and absorption algorithmso
F' (
Ab
sorp
tio
n)
oF' (Sigma) E (µmol photons m-2 s-1)o
F' ·
E
Emiliania huxleyi Emiliania huxleyi
Slope = 1.02R2 = 0.910
Sigma
Absorption
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Comparing the sigma and absorption algorithmso
F' (
Ab
sorp
tio
n)
oF' (Sigma)o
F'·
E
Oscillatoria sp. Oscillatoria sp.
Slope = 1.001R2 = 0.956
Sigma
Absorption
E (µmol photons m-2 s-1)
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Comparing the sigma and absorption algorithmso
F' (
Ab
sorp
tio
n)
oF' (Sigma)
Rhodomonas sp. Rhodomonas sp.
Slope = 0.893R2 = 0.903
Sigma
Absorption
E (µmol photons m-2 s-1)o
F' ·
E
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Instrument calibration
Oxborough et al. (2012):
RCII =𝐹𝑜𝜎PII
∙KR
𝐸LED
KR is an instrument specific constant, which cannot be used with other instruments of the same design
FastOcean calibration:
RCII =𝐹𝑜𝜎PII
∙ Ka
Ka is an instrument type-specific constant, which can be used with all FastOcean sensors
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Practical overview of the sigma method
Ambient FRRf
Dark FRRf
Instrument calibration –
Gain against chlorophyll a
ELED (photons m-2 s-1)
GPP estimated through –
JVPII = sPII'·[RCII]·(1 – C ) · E
Each measurement requires –
sPII' (m2) from ambient FRR-ST data
[RCII] (m-3) from chlorophyll (nPSII)
1 – C (proportion) = (Fm' – F')/(Fm' – Fo')
E (photons m-2 s-1) from a PAR sensor
Fm
Fo
Fm'
F'sPII
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Practical overview of the absorption method
Ambient FRRf
Dark FRRf
Fm
Fo
Fm'
F'
Instrument calibration –
Same as Sigma method plus:
Ka (m-1) = [RCII] · (sPII / Fo )
Derivation of Ka requires –
[RCII] (m-3) from flash O2 release
Fo (dimensionless) and…
sPII (m2) from dark FRRf data
GPP estimated through –
JVPII = aLHII · fPII' · EEach measurement requires –
aLHII (m-1) = ([Fm · Fo ] / [Fm – Fo ]) · Ka
fPII' = 1 – (F' / Fm' )
E (photons m-2 s-1) from a PAR sensor
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EJVPII = sPII' · [RCII] · (1 – C ) · E
RCII = Ka ∙Fo𝜎PII
𝜎PII′
1 − 𝐶 =Fm′ − F′
Fm′ − Fo′
Field measurements - sigma
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EJVPII = aLHII · fPII' · E
aLHII = ([Fm · Fo ] / [Fm – Fo ]) · Ka
fPII′ = 1 – (F′ / Fm′)
Field measurements - absorption
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Baseline fluorescence
ϕPII ≈ 0.9
RCII RCII + LHII
ϕPII ≈ 0.6
Fv
Fm≈ 0.5
Fv
Fm< 0.5 due to:
• Photoinactivation of RCII (photoinhibition)
• Downregulation (non-photochemical quenching)
FRRf
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Fb = Fm – Fv / (Fv/Fm)*
Where:• (Fv/Fm)* is the assumed 'true' Fv/Fm from
active RCII
• Fb is baseline fluorescence
Baseline fluorescence
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Fv / Fm = 0.5Fb = 0
Fv
Fm
Fo
Baseline fluorescence = 0
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Downregulation
Fv/Fm = 0.3Fb = 0
Fv
Fm
Fm
Fb
Photoinactivated RCII
Fv/Fm = 0.5Fb = 57% of Fo
Fo
Fb
Fv
Fo
Baseline fluorescence = 0 or Fb
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Fe-limited, oligotrophic conditions – natural phytoplankton population
Anna Macey, Tom Bibby and Mark Moore (University of Southampton, NOC)
[RCII] from D1
F o/s
PII
Fb = Fm – Fv / 0.45
[RCII] from D1
Fv/s
PII
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Fe-limited, oligotrophic conditions – natural phytoplankton population
𝐹𝑜
+Fe
0.6
0.45
a LHII
Fv/Fm = 0.45 gives the best fit to the +Fe points
Fv/Fm = 0.6 would give lower JVPII values than 0.45
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Summary
The absorption method:• Can be used to estimate JVPII on wide spatial and
temporal scales• Provides a much better S:N than the sigma method –
particularly at high ambient photon irradiance (E )• Baseline 'correction' of data looks to be a viable method
for dealing with low Fv/Fm from dark-adapted material
• More data are required to test this idea