Leaf Chamber Fluorometer ATP What is Fluorescence? Fluorescence - - PowerPoint PPT Presentation

6400-40 Leaf Chamber Fluorometer

ATP

What is Fluorescence?

• Fluorescence is light

emission by excited electrons decaying to the ground state

• Fluorescence is red

because the difference between S1 & ground state equals the energy of a photon of red light

ФF + ФD + ФP = 1 F + H + P = 1

Chlorophyll Fluorescence

F + H + P = 1 (eq. 1)

At satur urati ting ng light ht intensi tensity: No incre crease se in P with h further rther incre crease se in light ht intens ntensity y and F & & H maximum um F = Fm, H = Hm, P = 0 (eq. 2) Fm + Hm + 0 = 1 (eq. 3) Hm = 1 – Fm (eq.4 eq.4) If we as assum ume the rati tio of heat to fluorescenc rescence e de-exci cita tati tion n does not

• t change,

, H/F = Hm/Fm (eq. 5) H = F(1-Fm)/Fm (eq. 6) We can solve for H & & P if we m measure ure F in non-saturatin saturating light ht (F) an and satur urati ting ng light ht (Fm) P = 1-F-H (eq. 7) P =1 - F - [F(1-Fm)/Fm] (eq. 8)

P = Fm Fm-F/ F/ Fm Fm (eq.

• q. 9)

P = (Fm-F)/ Fm

' ' '

F F F F P

m m s m PSII light

F      

F F F F F P

m v m

• m

dark

  

 

leaf m s m leaf PSII

fI fI ETR

F F F

' '   

PSII/ CO2= mol e-/ mol CO2 fixed

PSII vs. CO2

• Theoretical minimum quantum requirement for non-cyclic

electron flow per CO2 fixed: 8 (C3), 12 (C4)

• Depends on proportion of products of electron transport used

for C assimilation relative to other processes (photorespiration, N2 & S2 metabolism)

C4 Maize

' ' Fm Fs Fm

PSII

  

leaf dark CO

I A A    

2

Applications of J

gm = mesophyll conductance Cc = [CO2] at site of carboxylation

gm

Constant J Method

• Use when J is constant over a range of

[CO2]

• ETR(J) from fluorescence
• Use * at the temperature
• A-Ci data solved for gm using statistical

method (Loreto et al., 1992)

Variable J Method

• A & Rd measured from gas exchange
• ETR (J) from fluorescence

Cc, Kc & Ko

• Cc can be

calculated from gm

• Vc,max (maximum

RUBP saturated rate of carboxylation) can be calculated from gm

𝐷𝑑 = 𝐷𝑗 − 𝐵 𝑕𝑛 𝑕𝑛 = 0.0045 𝑊

𝑑,𝑛𝑏𝑦

𝐵 = 1 − Γ ∗ 𝐷𝑗 𝑊

𝑑,𝑛𝑏𝑦 ∗ 𝐷𝑗

𝐷𝑗 + 𝐿𝑑 1 + 𝑃 𝐿𝑝 − 𝑆𝑒

qP and qN

' ' ' Fo Fm Fs Fm qP   

' ' Fo Fm Fm Fm qN   

Fm Fm Fm NPQ '  

Dark-Adapted Measurement

qN

qE: relaxes after a few minutes of darkness, as ΔpH dissipates and LHC converts from quenchers to funnels qT: relaxes after 10-20 minutes

• f darkness,

as LHC migrate from PSI back to PSII (“state transition”) qI: relaxes after hours, photoinhibition

LCF Design

• Red (630nm), Blue

(470nm), Far red (740nm) LED’s

• Fluorescence

Detection at 715nm

LCF Design

• 2 cm2 leaf area
• 0.4 kg
• Calibration information is

contained on-board

• Independent control of red and

blue LEDs for actinic light

Set-up Tips

Mea easur surement ement Sp Speci ecifics ics

Fluorescence Instrument Basics

• Higher fluorescence emission, better signal: noise
• Higher excitation intensities, higher fluorescence emission
• Higher excitation frequencies, higher excitation intensity
• Calculated parameters like Fv/Fm are not highly influenced by

fluorescence emission intensities (they are unitless)

• To compare across time, emission intensities do matter

Measuring Intensity

• Need to ensure that measuring

light is not actinic

• More of an issue for plants grown

at low light levels or photoinhibited

• Want a stable Fo without

increasing, decreasing, or “bumps”

• “Optimum Meas Intensity”

program in Light Source Calibration Menu

200 400 600 800 1000 1200 1400 1600 1800 2000 5 10 15 20 Time (s) Fluorescence

Ideal Not Ideal

Saturating Flash

• Make sure the flash is saturating

and stable

• Length: usually between 0.5 and

1 sec

• Intensity: selectable between 1-10
• “Optimum Flash Intensity”

program in Light Source Calibration Menu

F Time

Accurately estimate maximum fluorescence yield using Multiphase FlashTM Fluorescence methodology

Loriaux, S. D., T. J. Avenson, J. M. Welles, D. K. McDermitt, R. D. Eckles,B. Riensche and B. Genty. 2013. Closing in on maximum yield of chlorophyll fluorescence using a single multiphase flash of sub-saturating intensity. Plant, Cell &

• Environment. doi: 10.1111/pce.12115

Fm’

NPQ ETR ETR vs. AG gm PSII = (Fm’-F) Fm’ = (PSII*i**fII)

A Ci – Γ* [ETR + 8 (A + Rd)] ETR – 4 (A + Rd)

= (Fm-Fm’) Fm’ = Cc AN Vcmax climate modeling Cc

Used to measure Fm’ at infinite irradiance

Multiphase FlashTM fluorescence

AFm’

Fluorescence yield (F)

Irradiance (µE)

Phase 1 Phase 2 Phase 3

Irradiance (µmol m-2s-1)

AFm’

F (Phase 2)

1/Phase 2irradiance (m2 s mol-1) *104

10%

Extrapolated Fm’ ~ true Fm’

infinite irradiance

MPF increases the accuracy of Fm’, PSII and ETR (J) for field-grown plants

% difference ± SEa

Incident PPFD (µmol m-2 s-1)

n

Fm’ PSII, J

250

9

• 15.0

0 ±1.6

• 10.3 ±1.4

500

11

• 15.2 ±1.8
• 10.4 ±1.7

1000

14

• 19.0 ±1.9
• 18

18.5 ±2.0

1500

13

• 19.6 ±1.5
• 27.2

2 ±2.5

2000

14

• 16.2 ±1.6
• 29.9 ±3.3

Maize slope = 2.94 electrons/CO2

AFm’-derived J ○

slope = 4.7 electrons/CO2

EFm’-derived J

Fo or Fm Measurements

• Need sufficient dark-adaptation

time

• Pre-dawn best (must be

identical settings and identical position on leaf)

• Usually 20-30 min, but

sometimes not enough to fully relax qN (qI: can take hrs)

• Dark-adapting clips available

(#9964-091 $105 for 10 sets; 9964-092 $51 for 20 shutters)

• Can calculate photoinhibition from

difference in pre-dawn to “dark- adapted” measurements later in day

Fo’ Determination

Fo’ Relative to Fo

• Fo’ is the same as Fo when the

LHC, PSII centers, and the rest of the chain are at an identical state

• Fo’ is usually lower than Fo

because qN is not zero

• Fo’ can be higher than Fo if there

has been damage to PSII reaction center (heat* or chilling#)

*Schreiber and Bilger. 1987. In Tenhunen et al. (eds.)Plant Resp. Stress. Springer-Verlag, Berlin.

#van Kooten et. al. 1992. In: Murata (eds.) Res. In Photo. Kluwer, Dordrecht.

• Fo’ = Fo ((Fv Fm-1)+(Fo Fm’-1))-1
• Requires:
• All PS II centers open at Fo
• No change in regulation

between Fo and Fm

• No change in

photoinhibition between Fm’ and Fm

• Would usually measure Fo

and Fm after light-adapted measurement

*N. R. Baker and K. Oxborough. 2004. Chlorophyll fluorescence as a probe of photosynthetis productivity. In: G. C. Papageorgiou and Govindjee (eds.): Chlorophyll a Fluorescence: A signature of Photosynthesis. Pp 65-82. Springer, The Netherlands.

Alternative Fo’ Method

(*Baker and Oxborough, 2004)

• Far-red method potential

errors:

• qN may partly reverse

during far-red treatment

• Complete oxidation of QA

also relies on oxidation of PQ pool within a few seconds (and during ΔpH). Under these conditions, PQ

• xidation may be more rate-

limiting to electron flow than PS I excitation.

NPQ vs. qN?

• Excitation energy transfer in

the light should be measured as Fv’/Fm’ if one can accurately measure Fo’

• The decrease in Fv’/Fm’ in the

light is caused by increased thermal dissipation in LHCII with increasing light, so non- photochemical quenching parameter should correlate fairly linearly with Fv’/Fm’, which is not always the case

• The Stern-Volmer equation:

NPQ = (Fm-Fm’) (Fm’)-1

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

0.2 0.4 0.6 0.8 1

qN NPQ Fv'/ Fm'

NPQ qN

Fluorescence Light Curves

• To minimize risk of

photoinhibition:

• Order low to high (slow)
• Order intermediate, low,

intermediate, high (quicker)

• Order intermediate, high

(quickly), intermediate, low (quickest)

• To check: measure Fo & Fm

20-30 min after dark-adapting & compare to original Fo and Fm before curve

• Same environmental control

constraints as gas exchange response curves

A few considerations for comparing Gas Exchange and Fluorescence

• Depends on proportion of products of electron transport used for C

assimilation relative to other processes (photorespiration, N2 & S2 metabolism)

• αleaf for red is typically 0.87 and blue is 0.90, but can vary between

species and treatments

• To measure, must use an integrating sphere
• LED wavelengths may be preferentially absorbed in the upper layers of

the leaf, while gas exchange is measured from the entire leaf

Fm’- Fs = F = PSII Fm’ Fm’ A- Adark = CO2 I leaf

Garbage in = Garbage out

• Just because the parameter is listed on the display and in the data file,

that does not guarantee it is meaningful

• The fluorometer cannot determine whether all of the data was collected

appropriately