using fast repetition rate fluorometry to estimate psii electron flux per unit volume
DESCRIPTION
Chelsea's Chief Scientist, Dr Kevin Oxborough gave a presentation at an Invitational seminar at the Max Planck Institute for Marine Microbiology on 12 August 2014. The presentation was entitled "Using fast repetition rate fluorometry to estimate PSII electron flux per unit volume: A purely optical method for estimating GPP?” http://www.mpi-bremen.de/en/Home.htmlTRANSCRIPT
Using Fast Repetition Rate
fluorometry to estimate PSII electron
flux per unit volume: A purely optical method for estimating GPP?
Kevin Oxborough
Principal Scientist
Kevin Oxborough (CTG)
Mark Moore (Southampton)
Dave Suggett, Richard Geider (Essex)
H2O
PQ
e-
PC
NADP
e-
PSII PSIb6f
CO2 assimilation
e-
PC
PQH2
Photosynthetic electron transport
e-PSII flux
FRRfhn hn
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
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
Closed RCII
Photochemistry and fluorescence
f
d
H2O
PQ
ϕ𝑓 =𝑘𝑓
𝑘𝑓 + 𝑘𝑑
ϕ𝑝 = 0
The FRRf technique
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
Saturation phase100 x 1 µs flashlets
2 µs pitch
Relaxation phase40 x 1 µs flashlets
50 µs pitch
Saturation and relaxation phases
100 x 1 µs flashlets
2 µs pitch
40 x 1 µs flashlets
50 µs pitch
Saturation and relaxation phases
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)
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
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
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
Connectivity among RCIIs
Separate Package
Open RCII
Closed RCII
LHII
Lake
Open RCII
Closed RCII
LHII
Partial connectivity
Dimer model
Connectivity among RCIIs
Photosystem II dimer
taken from Nield & Barber (2006)
• X-ray crystallography• Core from cyanobacterium• LHCII from spinach
• Cryo-EM from spinach
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′
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
JVPII = 𝑎PII′ ∙ 𝐸
FRRf FRRf
JPII = 𝜎PII′ ∙ 𝐸
From PSII electron flux to GPP
= 𝑎LHII ∙ 𝜙PII′ ∙ 𝐸Sigma method
Absorption method
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)
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
The absorption method (Oxborough et al. 2012)
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
Consequence:
RCII =𝐹𝑜𝜎PII
∙KR
𝐸LED
Calculation of [RCII] using the absorption method
Hypothesis:
𝑘𝑝
𝑘𝑓falls within a very narrow range
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
[RCII] x 1016 m-3
F o/ s
PII
NIOZ PROTOOL workshop, Yerseke (2012)
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]
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
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
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
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)
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
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
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
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
EJVPII = sPII' · [RCII] · (1 – C ) · E
RCII = Ka ∙Fo𝜎PII
𝜎PII′
1 − 𝐶 =Fm′ − F′
Fm′ − Fo′
Field measurements - sigma
EJVPII = aLHII · fPII' · E
aLHII = ([Fm · Fo ] / [Fm – Fo ]) · Ka
fPII′ = 1 – (F′ / Fm′)
Field measurements - absorption
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
Fb = Fm – Fv / (Fv/Fm)*
Where:• (Fv/Fm)* is the assumed 'true' Fv/Fm from
active RCII
• Fb is baseline fluorescence
Baseline fluorescence
Fv / Fm = 0.5Fb = 0
Fv
Fm
Fo
Baseline fluorescence = 0
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
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
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
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