Sample preparation
HealthyZ. marina with intact rhizomes-systems was harvested on the sea floor at around 3 m depth near Rongcheng (37º 16’N, 122º 41’E) in the Weihai, Shandong province, China. Samples were precultured for 3 days in an aquarium under conditions of 15 °C and a 10: 14 light: dark cycle with a minimum saturation light intensity of 100 µmol photons m−2s−1. Prior to each experiment, each shoot was “standardized” to similar leaf morphology. Leaves for experimental measurement were collected from 2 cm above the sheath to keep the same age.
Experimental treatment
Prior to experimental treatment, pre-cultured Z. marina plants were dark-adapted overnight. To obtain the photoinhibition rate constant, Z. marina leaves were pre-incubated with lincomycin for 3 h in the dark and were then exposed to 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 µmol photons m-2 s-1 (µE) at 15 °C (Miyata et al., 2012).Z. marina leaves, which were dark-adapted and exposed to 300 µE for 3 h at 15 °C, were used for1O2contents, AsA levels and chlorophyll a fluorescence measurement, and proteome analysis. The light intensity of 300 µE was approximated the maximum midday light intensity at the site of collection. The home-built LED lamps with color temperature of 6000 K and adjustable illumination intensity up to 1000 µmol photons m−2 s−1 were used for the sample preculture and light treatments. The spectrum of the home-built 6000 K LED lamp measured by HR-450 (HiPoint, China, Taiwan) has the main emission bands at around 450 nm (Fig. S2), similar with the spectrum of the 3 m depth under seawater where the plants are naturally located (Kirk, 2010; Olsen et al., 2016).
Due to lack of genetic operating system, the physiological roles of AsA and PSII-CEF were examined using inhibitors. PSII-CEF was inhibited or enhanced via incubation of 2,5-dimethyl-p-benzoquinone (DMBQ, TCI, Tokyo, Japan) at the concentration of 125 or 62 µM, respectively (Ananyev et al., 2016, 2017). AsA synthesis were inhibited via 50 µM rotenone purchased from Sigma-Aldrich (Millar et al., 2003; Garmier et al., 2008). Although the chloroplast NADPH dehydrogenase-like complex was also sensitive to rotenone (Garmier et al., 2008), no significant side effect of 50 µM rotenone was observed on PSI-CEF of Z. marina showed by the initial rate of P700+re-reduction (V re-red, Fig. S3). To test the roles of AsA and PSII-CEF, four different combinations of AsA and PSII-CEF inhibitions were conducted: Light, Light + Rotenone, Light + DMBQ, and Light + Rotenone + DMBQ, forming the base of a factorial experimental design. Prior to exposure,Z. marina leaves saturated with filtered seawater or with the seawater solution containing inhibitors were incubated in the dark for 10 min at 15 °C. Moreover, leaves treated with 125 µM DMBQ or 50 µM rotenone under darkness for 190 min showed no significant changes inF v/F m (Fig. S4). A stock solution of 10 mM DMBQ and 25 mM rotenone was prepared with dimethyl sulfoxide whose concentration used in this study had no significant effect on Z. marina as indicated byF v/F m (Fig. S5).