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).