Presentation on theme: "B P1 P2 P3 Fm Fo Fm’ A Fo’act. ≈ Fo’calc. 2 min 3 min qPd = (Fm’-Fo’ act. )/(Fm’-Fo’ calc. ) Fo’ actual Fo’ calculated Fm’ AL FR (10s) 90 190 285 425 625."— Presentation transcript:
B P1 P2 P3 Fm Fo Fm’ A Fo’act. ≈ Fo’calc. 2 min 3 min qPd = (Fm’-Fo’ act. )/(Fm’-Fo’ calc. ) Fo’ actual Fo’ calculated Fm’ AL FR (10s) 90 190 285 425 625 820 1150 1500 625 820 1150 SP Fig. S1 - Ware et al. Fig. S1 a A typical high light acclimated plant chlorophyll fluorescence scheme of induction with an eight step increasing actinic light (AL) routine. In this example 0, 90, 190, 280, 420, 625, 820, 1150, 1500 µmol m -2 s -1 AL intensities were used. 80 and 90% AL intensities of the aforesaid values were achieved by carefully extracting the fibre- optic from the emitting diode and determining the AL intensity with a Walz MQS-B sensor. This allowed a more accurate reflection of PSII susceptibility to photodamage to be realised. For detailed explanation of routine development see Ruban & Belgio (2014). b A zoomed in region of the fluorescence scheme (a) illustrating the timing and application of 625, 820 and 115 µmol m -2 s -1 AL (upward arrow and downward arrow demonstrate the turning of AL on and off respectively), along with saturating pulses (SP) (P1, P2, P3). P1 indicates an SP in the dark, or after 10 sec oif far red (FR) light, P2 during AL illumination, and P3 at the end of AL. The difference between Fo’act. and Fo’calc. is determined at P1, and subsequently used to calculate qPd. At low AL intensities there is little to no difference between Fo'calc. and Fo'act., but under increasing AL intensities the two values diverge. See also ‘Materials and methods’ for a detailed description. The timing scheme in the dark was: (AL off)(FR on)-(10 s)-(FR off/SP)-(5 s)-(AL on).
(a) (b) ML Fig. S2 - Ware et al. Fig. S2a ΔFo’ [(Fo’act.-Fo’calc.)/Fo’act.] results obtained from fluorescence traces for each AL intensity were averaged for medium light grown plants. Error bars show SEM (n = 10). b Relationship between NPQ and qPd (open circles) and NPQ and PSII actual yield (closed circles) for medium light grown plants. Data points were averaged from 30 repeats on whole intact leaves. Error bars show the standard error of the mean (n = 30). The theoretical yield (continuous line) was calculated using equation 1 of ‘Materials and methods’, but with qPd always equal to= 1.
(a) LL (b) ML (c) HL Fig. S3 - Ware et al. Fig. S3 Absorbance spectra (Hitachi, U-3310 spectrophotometer) conducted on bands obtained from sucrose gradients (Fig. 4) for (a) low light (b) medium light (c) high light grown plants. All bands were normalised to 0 at 750 nm. Band 2-6 correspond to monomers, trimeric LHCII, LHCII-CP29-CP24, PSII core complexes respectively. Bands 1 and 7 were measured and recorded as free pigments and PSI-LHCI accordingly (data not shown).
Fig. S4 - Ware et al. Fig. S4 An example band absorbance spectra from Fig. S3, with traces again zeroed at 750 nm. Using OriginPro 9.0, the mathematical area under each trace was calculated using the integrate function between 550 and 750 nm. This area was used to calculate the total amount of arbitrary chlorophyll. This was achieved by multiplying the total amount of the band extraction from the sucrose gradient (ml) by the dilution factor of solution used to perform the absorbance spectra, and by the area measured under the trace. This arbitrary chlorophyll value was divided by the number of chlorophylls per complex in each band (42 chlorophylls per LHCII trimer (band 3), 112 per LHCII- CP29-CP24 complex (band 4) and 35 per PSII core complex (band 5)) to ascertain the amount of complexes present.
Fig. S5 PSII fast fluorescence induction traces performed on detached lincomycin treated leaves. Vacuum infiltration with 30 μM DCMU was perfomed 20 sec before exposure to 7 μmol m-2 s-1. Traces are the mean values for 3 repeats. All traces were zeroed at Fo and normalised to 1 at Fm.
Fo’ calc. het. = 1/(1/(0.96*0.2+0.04*1) – 1 + (1/(0.96/(NPQ+1) + 0.04/(2*NPQ+1)))) Fo’ calc. het. = 1/(1/(n*Fo+(1-n)*Fm) – 1/Fm + (1/(n/(NPQ+1) + (1-n)/(2*NPQ+1)))) For the low light case : Fo’ calc. = 1/(1/Fo – 1/Fm + 1/Fm’) Fm’ = Fm/(NPQ+1) Fo’ calc. = 1/(1/Fo – 1/Fm + (NPQ+1)/Fm) where Fm=1; Fo ideal = 0.2; 3% detached antenna; NPQ in detached = 2*NPQ where (1-n) is the fraction of detached LHCII n = (5 – Fo ex /Fo ideal )/4 Fig. S5 - Ware et al. Fig. S6 Association of NPQ and Fo’act. recorded during a gradually increasing actinic light routine performed on whole intact leaves from low light grown plants. Error bars show the standard error of the mean (n = 30). The continuous black line was plotted using the formula of Oxborough and Baker (Materials and methods, equation 3). The continuous red line was plotted using a modified formula for a heterogeneous photosynthetic membrane system (Results and discussion, equation 7).