Chlorophyll Fluorescence Imaging Protocol

Chlorophyll fluorescence imaging was performed using a customised FluorCam imaging fluorometer fitted with a white and red LED panel (Additional file 1: Fig. S1). Shutter time and sensitivity of the charge-coupled device (CCD) were adjusted in accordance with sample. The FluorCam is located in a temperature controlled dark room maintained between 20 and 22 °C.
Modern fluorometers commonly use a modulated light source at a known frequency to induce chlorophyll fluorescence—otherwise known as pulse-amplitude modulated (PAM) fluorescence—where the detector is set to measure at the same frequency as the excitation [37 (link)]. This methodology allows measurements to occur when the plant is illuminated. During a typical measurement, the plant is dark adapted (between 20 and 60 min) to allow maximal plastoquinone A (Q_A) oxidation after which the leaf is exposed to a saturating flash of light that maximally reduces Q_A, closing all PSII reaction centres. This procedure gives a maximum fluorescence value (F_m) and, in the light, allows the separation of the photochemical (e.g. PSII operating efficiency—F_q′/F_m′) and non-photochemical (e.g. Non-photochemical quenching—NPQ) processes in the leaf under specific photosynthetic photon flux density (PPFD) conditions. The parameter F_q′/F_m′, also termed ɸPSII or quantum yield (QY), is a measure of the proportion of absorbed light utilised by PSII and therefore can also be used, in combination with measurements of leaf absorbance, to calculate linear electron transport rate (ETR). These parameters (Table 1) are key in the identification of differences between different lines, treatments (biotic or abiotic) or genotypes [20 (link)–22 (link), 38 (link), 39 ]. Many instruments are available for assessment of these parameters either as spot measurements or as images. The benefit of imaging chlorophyll fluorescence is the ability to analyse both temporal and spatial variation in PSII efficiency [28 (link)].Table 1

Commonly used abbreviations and equations employed when measuring chlorophyll fluorescence

Parameter	Formula	Definition
F, F′, Fs′	n/a	Steady state fluorescence emission from dark- or light-adapted (‘) leaf, respectively. F′ is sometimes referred to as Fs′ when at steady state.
Fm, Fm′	n/a	Maximal chlorophyll fluorescence measured in a dark- or light-adapted state respectively
Fo, Fo′	n/a	Minimal chlorophyll fluorescence measured in a dark- or light-adapted state respectively
Fv, Fv′	n/a	Variable chlorophyll fluorescence measured as the difference between dark- or light-adapted Fm/Fm′ and Fo/Fo′.
Fv/Fm	(Fm–Fo)/Fm	Maximum quantum efficiency of PSII.
Fv′/Fm′	(Fm′–Fo′)/Fm′	Maximum efficiency of PSII in the light.
Fq′/Fm′	(Fm′–F′)/Fm′	PSII operating efficiency: the quantum efficiency of PSII electron transport in the light. AKA ΦPSII, quantum yield or ΔF/Fm′
ETR or J	ΦPSII (AKA Fq′/Fm′) × PPFDa × (0.5)	Linear electron transport rate; where PPFDa is absorbed light (μmol m−2 s−1) and 0.5 is a factor that accounts for the partitioning of energy between PSII and PSI.
NPQ	(Fm–Fm′)/Fm′	Non-photochemical quenching: estimates the rate constant for heat loss from PSII.
qL	(Fq′/Fv′)/(Fo′/F′)	Estimates the fraction of open PSII centers (QA oxidized); considered a more accurate indicator of the PSII redox state than qP
qP	(Fm′–F′)/(Fm′–Fo′) AKA Fq′/Fv′	Photochemical quenching: relates PSII maximum efficiency to operating efficiency. Non-linearly related to proportion of PSII centers that are open. 1–qP has also been used to denote proportion of closed centers

A summary table of the commonly used chlorophyll fluorescence parameters and corresponding equations. For a more comprehensive review please refer to Murchie and Lawson [21 (link)], Baker [20 (link)] and Maxwell and Johnson [22 (link)]