Light Adaptation

Imaging of chlorophyll a fluorescence was performed using FluorCam imaging fluorimeters (Photon Systems Instruments, Brno, Czech Republic). Shutter time and sensitivity of the charge-coupled device (CCD) camera (SN_FC800) were adapted to the particular object. Measurements of Φ_PSII (see above) were made after light adaptation of the plants in adaptation tunnel and subsequent to an illumination period in the FluorCam-chamber as indicated in the results section. Duration of the saturating light pulse to induce F_m′ was 800 ms with an intensity of 4100 µmol m⁻² s⁻¹ (white light) in the system for small plants and 3800 µmol m⁻² s⁻¹ in the large system, respectively. Light response curves of the photosynthetic electron transport of PSII (ETR) were obtained by a stepwise decrease of the actinic light intensity (duration of each step 60 s). The apparent rate of ETR was determined as ETR=Φ_PSII × PAR × 0.5 × 0.84 where 0.5 is a factor that accounts for the fraction of excitation energy distributed to PSII and the factor 0.84 corresponds to the leaf absorbance. Both factors are empirical mean factors. Therefore the results of the ETR calculations are considered as apparent.

The method for studying mouse rod dark adaptation in vivo using LKC® ERG system had been described in detail previously13 (link). Briefly, dark-adapted animals were anesthetized with ketamine/xylazine cocktail (100/20 mg/kg) by intraperitoneal injection. The pupils of the anesthetized animals were dilated with a drop of 1% atropine sulfate solution and the animals were transferred to a 37 °C heating pad with a feedback anal thermal probe. The reference electrode was inserted subcutaneously beneath the scalp and 2.5% Gonak hypromellose ophthalmic demulcent solution was applied to the cornea. A contact lens electrode was positioned on the cornea of each eye to detect electrical signals from retina. Excessive Gonak solution was removed from the eyes with tissue paper and the animals were allowed to stabilize in darkness for 15 minutes before beginning the recordings. Test flashes from a 530 nm LED, ranging from 2.5 × 10⁻⁵ cds m⁻² to the 25 cds m⁻² limit, were used to elicit photoresponses from each eye, and white Xenon flashes were used to produce saturated photoresponses. Sufficient time was allowed between individual test flashes to allow full recovery of the retina and avoid gradual response run-down due to light adaptation. For dark adaptation testing, a bright green (505 nm) LED light was applied to both eyes for 30 seconds to photobleach an estimated 90% of the visual pigment. The recovery of the ERG responses was monitored at fixed post-bleach time points within 2 hours after the bleach. The maximal response amplitude, r_max, was recorded at the brightest light intensity, and S_f was estimated as the ratio of dim flash response amplitude and the corresponding flash intensity in the linear range of the intensity-response curve, about 20% to 30% of the maximum. The post-bleach maximal amplitude (r_max) and sensitivity (S_f) were normalized to their dark adapted pre-bleach level, r^DA_max and S_f^DA, respectively.

Data were analyzed using Clampfit 10.7 (Molecular Devices), Microsoft Excel and Origin 9.8.5 (64 bit, SR2, OriginLab) and presented as mean ± SEM p-values of <0.05 (Student’s t test) were considered significant. The intensity-response relationships data were fitted by a hyperbolic Naka–Rushton function using the following equation:
$$\frac{R}{R_{max}}=\frac{I^n}{I^n+I_{1\mathrm{∕}2}^n},$$ where R is the flash response, R_max is the maximum response amplitude, I is the flash intensity, n is the Hill coefficient, and I_1/2 is the intensity to produce half-saturating response. The light adaptation data were fitted by a modified Weber–Fechner function, as follows:
$$\frac{S_f}{S_{fDA}}=\frac{I_0^n}{I_0^n\hspace{0.17em}+\hspace{0.17em}{I}^n},$$ where S_f is the rod sensitivity (as defined above), S_f^DA is the rod sensitivity in darkness, n is a slope factor (Hill coefficient), I is the background light intensity (in photons μm⁻² s⁻¹), and I₀ is the background light intensity needed to reduce the sensitivity to 50% of that in darkness.

The above-mentioned potted 6-month-old seedlings with 5 cm heights were selected and moved to a dark room overnight at 25 ^◦C. Five plants (one plant in a pot) with fully open top leaves were used for ChlF measurements. In addition, the upper fully expanded leaves of 2.4-year-old seedlings were collected from July to August 2021 for the following LI experiments. Five plants (one leaf per plant) per light treatment were used for ChlF measurements. The surfaces of the leaves were illuminated with 50, 100, 300, 500, 1000, 1500, and 2000 μmol m^–2 s^–1 PPFD using a portable pulse amplitude-modulated fluorometer (PAM-2000, HeinzWalz, Effeltrich, Germany). Dark-adapted plants were exposed to light stepwise from low to high levels of PPFD, and ChlF parameters were measured during 60 min of irradiation and dark adaptation for 30 min. individual data points were recorded at 2 min intervals over a 90 min period, followed by calculating the parameters below. Seven gradients of photometry were used to measure two ages of M. oiwakensis, but more detailed light adaptation assessments were performed due to little differences in parameters observed in the light curve (Additional file 1: Fig. S3).
The potential quantum efficiency of PSII (Fv/Fm) was calculated from (Fm - Fo) / Fm (Demmig-Adams et al. 1996 (link)). The actual PSII efficiency (ΔF / Fm’) is the effective quantum yield of linear electron flux through PSII, and used to express the ability of PSII to perform photochemistry. Values of the minimal ChlF (Fo) and maximal ChlF (Fm) of dark-adapted samples were determined using modulated irradiation of a weak light-emitting diode beam (measuring light) and saturating pulse, respectively. Fm’ is the maximal fluorescence during illumination determined by applying a saturating flash. Measured leaves were dark-adapted for 30 min before performing light-inducing runs. The photochemical ΦPSII was calculated as (Fm’ - Ft)/Fm’, where Ft is the steady-state fluorescence at each PPFD level (Maxwell and Johnson 2000 (link)). Furthermore, the degree of photo-inhibition is calculated as 100% minus the relative value of F_v/F_m after 30 min of dark adaptation, where the F_v/F_m value of the same leaves before illumination is considered to be 100%. The apparent rate of the photosynthetic electron transport rate (ETR) of PSII was obtained as ETR = ΔF/F_m′ × PPFD × 0.5 × α, where the factor 0.5 assumes equal excitation of both PSII and PSI; α is leaf absorption, and we used the mean “default” value of 0.84 for green leaves (Björkman and Demmig 1987 (link); Lin et al. 2020 (link)). The following effective quantum yields were measured using the instant light-response curve program. From these data, several parameters can be computed based on modulated fluorescence kinetics. Degree of photoinhibition (photo-inhibition %) = 100%—relative value of Fv/Fm after 30 min of dark adaptation (Fv/Fm value of same leaves before illumination as 100%). The NPQ coefficient and its components: NPQ = (Fm—Fm’) / Fm’ (Müller et al. 2001 (link); Weng et al. 2011 (link)). Energy-dependent quenching (qE) of NPQ is a mean of rapidly quenching energy, which is calculated as (Fm dark (2 min)—Fm’60 min) / Fm’60 min = (F_mD₂—F_m60′) / F_m60′ (Johnson and Ruban 2011 (link)). However, photo-inhibitory quenching (qI) is NPQ due to decreased CO₂ fixation, which is calculated as [Fm—Fm dark (60 min)] / Fm’30 min = (F_m—F_mD₃₀) / F_m60′ (Müller et al. 2001 (link)). In addition, the part after the reaction of qE is (qZ + qT), and calculated as (F_mD_30—F_mD₂) / F_m60′ (Maxwell and Johnson 2000 (link); Nilkens et al. 2010 (link)). The F_m60′ is the maximum fluorescence value of leaves at 60 min of light exposure. Both F_mD₂ and F_mD₃₀ are the F_m values measured at 2 and 30 min, respectively, after dark recovery (Müller et al. 2001 (link); Wang et al. 2022 (link)). Measurements were recorded with WinControl-3 software (Heinz Walz).