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

Chlorophyll Fluorescence Ф F + Ф D + Ф P = 1 F + H + P = 1

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 (eq. 2) F = Fm, H = Hm, P = 0 (eq. 3) Fm + Hm + 0 = 1 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 ot change, , (eq. 5) H/F = Hm/Fm (eq. 6) H = F(1-Fm)/Fm 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 ) (eq. 7) P = 1-F-H (eq. 8) P =1 - F - [F(1-Fm)/Fm] P = Fm Fm-F/ F/ Fm Fm (eq. q. 9)

P = (Fm-F)/ Fm  F F F   m o v P dark F F m m   F ' F F     m s P PSII light F F ' ' m m '  F F      m s ETR fI fI PSII leaf leaf F ' m

 PSII vs.  CO2  Fm ' Fs   C 4 Maize PSII Fm '  A A   dark  CO 2 I leaf  PSII/  CO2= mol e - / mol CO 2 fixed • 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, N 2 & S 2 metabolism )

Applications of J g m = mesophyll conductance C c = [CO 2 ] at site of carboxylation

g m Constant J Method • Use when J is constant over a range of [CO 2 ] • ETR(J) from fluorescence • Use  * at the temperature • A-Ci data solved for g m using statistical method (Loreto et al., 1992) Variable J Method • A & Rd measured from gas exchange • ETR (J) from fluorescence

C c , K c & K o • Cc can be 𝐷 𝑑 = 𝐷 𝑗 − 𝐵 calculated from gm 𝑕 𝑛 • Vc,max (maximum RUBP saturated 𝑕 𝑛 = 0.0045 𝑊 𝑑,𝑛𝑏𝑦 rate of 1 − Γ ∗ 𝑊 𝑑,𝑛𝑏𝑦 ∗ 𝐷 𝑗 carboxylation) can 𝐵 = − 𝑆 𝑒 be calculated from 𝐷 𝑗 + 𝐿 𝑑 1 + 𝑃 𝐷 𝑗 gm 𝐿 𝑝

q P and q N  Fm ' Fs  q P  Fm ' Fo '  ' Fm Fm  q N  Fm Fo '  Fm Fm '  NPQ Fm Dark-Adapted Measurement

q N q E : relaxes after a few minutes of darkness, as Δ pH dissipates and LHC converts from quenchers to funnels q T : relaxes after 10-20 minutes of darkness, as LHC migrate from PSI back to PSII (“state transition”) q I : 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

Mea easur surement ement Sp Speci ecifics ics Set-up Tips

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 2000 light is not actinic Ideal 1800 Not Ideal • More of an issue for plants grown 1600 1400 at low light levels or photoinhibited Fluorescence 1200 • Want a stable Fo without 1000 increasing, decreasing, or “bumps” 800 600 •“Optimum Meas Intensity” 400 program in Light Source 200 Calibration Menu 0 0 5 10 15 20 Time (s)

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” F program in Light Source Calibration Menu Time

Accurately estimate maximum fluorescence yield using Multiphase Flash TM 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’ -F)  PSII (Fm- Fm’ ) = NPQ Fm’ Fm’ Fm’ = (  PSII *i*  *f II ) ETR g m A = C i – Γ * [ETR + 8 (A + R d )] ETR – 4 (A + R d ) ETR vs. A G climate C c A N V c max modeling C c

Multiphase Flash TM fluorescence Extrapolated Fm’ ~ true Fm’ Phase 1 Phase 2 Phase 3 Irradiance (µE) Fluorescence yield (  F ) 10% Irradiance (µmol m -2 s -1 ) A Fm ’  F (Phase 2) A Fm ’ infinite irradiance 1/Phase 2 irradiance (m 2 s mol -1 ) *10 4 Used to measure Fm’ at infinite irradiance

MPF increases the accuracy of Fm ’,  PSII and ETR (J) for field-grown plants % difference ± SE a E Fm ’ -derived J Incident slope = 4.7 electrons/CO 2 PPFD  PSII , J Fm ’ n (µmol m -2 s -1 ) 9 -15.0 0 ±1.6 -10.3 ±1.4 250 500 11 -15.2 ±1.8 -10.4 ±1.7 slope = 2.94 electrons/CO 2 A Fm ’ -derived J ○ 14 -19.0 ±1.9 -18 18.5 ±2.0 1000 Maize 1500 13 -19.6 ±1.5 -27.2 2 ±2.5 14 -16.2 ±1.6 -29.9 ±3.3 2000

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 q N (q I : 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 q N 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.

Alternative Fo ’ Method (*Baker and Oxborough, 2004) • Far-red method potential • Fo ’ = Fo ((Fv Fm -1 )+(Fo Fm’ -1 )) -1 • Requires: errors: • q N may partly reverse • All PS II centers open at Fo during far-red treatment • No change in regulation • Complete oxidation of Q A between Fo and Fm also relies on oxidation of • No change in PQ pool within a few photoinhibition between Fm ’ seconds (and during Δ pH). and Fm Under these conditions, PQ • Would usually measure Fo oxidation may be more rate- and Fm after light-adapted limiting to electron flow than measurement PS I excitation. *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.

NPQ vs. q N ? • Excitation energy transfer in 4.5 1 the light should be measured as NPQ 0.9 4.0 Fv ’/ Fm ’ if one can accurately 0.8 qN 3.5 measure Fo ’ 0.7 3.0 0.6 qN • The decrease in Fv ’/ Fm ’ in the NPQ 2.5 0.5 2.0 light is caused by increased 0.4 1.5 0.3 thermal dissipation in LHCII 1.0 0.2 with increasing light, so non- 0.5 0.1 photochemical quenching 0.0 0 0 0.2 0.4 0.6 0.8 1 parameter should correlate Fv'/ Fm' fairly linearly with Fv ’/ Fm ’, which is not always the case • The Stern-Volmer equation: NPQ = (Fm-Fm ’) ( Fm ’) -1

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

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