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).