Absorption spectrum and oxygen evolution measurements
To monitor the contents of AsA which could directly protect photosystems, AsA levels in chloroplasts were monitored by using vitamin C assay kit (A009, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).1O2in chloroplasts contents were measured by using plant ROS singlet oxygen assay kit (GMS10150.4, GENMED, Boston, MA, USA). Chloroplasts were isolated from Z. marina leaves, which were dark-adapted and exposed to 300 µE for 3 h, by using the plants leaf chloroplast rude divide kit (GMS16004.1, GENMED). To monitor change in carbon fixation ability during 300 µE light exposure, the Rubisco carboxylase activity was determined by using ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity detection kit (BC0440, Solarbio, Beijing, China). Absorption measurements were performed using a multifunctional enzyme-labeled instrument (Tecan, Männedorf, Switzerland). O2evolution rate used to reflect the leaves photosynthetic rate was measured at 15 °C with a liquid-phase oxygen electrode system (Chlorolab2+, Hansatech, Norfolk, UK).
Chlorophyll a fluorescence and 820 nm modulated reflection (MR820nm) measurements
A pulse-amplitude modulated fluorometer (Mini-PAM; Walz, Germany) was used to measure NPQ. During the course of measurements, the PAM fiber was placed in a fixed position at 60° in relation to Z. marina leaves to avoid shading or darkening.
Multi-function plant efficiency analyzer 2 (M-PEA-2; Hansatech, Hercules, UK) was used to simultaneously detect the kinetics of prompt fluorescence (PF), delayed fluorescence (DF), and MR820nm and thus to monitor photochemical activity and dissect events in the electron transfer chain (Strasser et al., 2010; Gururani et al., 2015)10,62. Changes in the OJIP fluorescence rise kinetics were assessed by calculating the difference value of variable fluorescence curves as ΔV t = Δ[(F t - F O) / (F m - F O)]. The relative variable fluorescence at the K-step (W K) was an indicator of the PSII electron donation capacity (Strasser, 1997; Brestic et al., 2012){Brestic, 2012 #265;Strasser, 1997 #269}. The decay kinetics of DF at maximum I 1 were fitted with the function DF(t ) = L 1 × exp(-t /τ 1) + L 2 × exp(-t /τ 2) + L 3, whereL 1 and L 2represent the amplitudes of the decay kinetics component and τ 1 andτ 2 represent their lifetimes (in ms), respectively. L 1 corresponded to the electron transport capacity at PSII donor side (Goltsev et al., 2009; Gao et al., 2013). MR820nm obtained by a modulated measuring light at 820 nm was measured after far-red light was closed (Zhang et al., 2011).V re-red, the initial rate of P700+re-reduction, was calculated to quantify PSI-CEF activity. Detailed calculations and physiological explanations of parameters are listed in Table S2.