using fast repetition rate fluorometry to estimate psii electron flux per unit volume

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