Damaging mechanism
Although the reduction of QA represented by 1 -V J was
significantly increased, the reduction degree of QA was
low (by ~4.5%) (Fig. 4A).
Consistently, no significant
increases in chloroplast1O2 content and, the quencher of1O2,
GPX were observed following light exposure (Fig. 4A and Table
S1), while
PsbH,
which stabilized the combination
of PSII and β-carotene associated
with scavenging 1O2, was significantly
downregulated (Table S1).
Nevertheless, PSII photochemical
activity significantly decreased, indicated by a decrease
in F P and an
increase in F O following light exposure (Fig.
4B). This suggested the existence of an additional source of damage
other than 1O2.
Photoprotective mechanisms
l-galactono-1,4-lactone dehydrogenase (GLDH),
the terminal enzyme in the major
Smirnoff-Wheeler pathway for AsA biosynthesis, together with the
AsA level in the chloroplast were
significantly upregulated after light exposure (Fig. 5A), providing a
clue to AsA being the alternative electron
donor. Additionally, analysis of
the K step in ΔV t curves andF P in OJIP curves showed: there was the lower
PSII
electron
donation capacity and PSII photochemical activity induced by 125 μM DMBQ
binding at the PQ site to uncouple PSII-CEF, compared to 62 μM DMBQ
binding at the QBsite to enhance PSII-CEF (Fig. 5B). This suggested that PSII-CEF serves
as a possible alternative electron donation pathway.
To
test the roles of
AsA
and PSII-CEF in safeguarding PSII
function, a factorial design experiment with different combinations of
rotenone and DMBQ, the inhibitors
of AsA and PSII-CEF, was conducted.
Compared withF v/F m values,
the order of PSII activity was:
Light > Light + Rotenone > Light + DMBQ
> Light + Rotenone +
DMBQ (Fig. 6A).
Simultaneously, inhibition of AsA
synthesis or uncoupling of PSII-CEF led to the further decreased PSII
electron donation capacity indicated by W K, and
the decrease was more obvious during the late illumination (Fig. 6A).
Furthermore, Western-blotting analyses showed no visible changes of D1,
CP43 proteins after light exposure and their degradation was detected
after both inhibitor treatments, indicating that
AsA and PSII-CEF prevented the
damage of PSII RC via donating electrons (Fig. 6B). The suppression of
temporary electron donor and the alternative electron flow also led to a
further impairment of OEC, as characterized by the distinct degradation
of PsbO and PsbP proteins (Fig. 6B).