Mechanisms By Which The Infection Of Sclerotinia Sclerotiorum(Lib.) De Bary Affects The Photosynthetic Performance In Tobacco Leaves

Sclerotinia sclerotiorum (Lib.) de Bary (S. sclerotiorum), the host range and geographical distribution of which is widespread, belongs to asconycete subdivision. The damage of photosynthetic apparatus and the decrease of photosynthetic rate is the direct reason for the reduction of crop yields. Though much work related to the interaction between S. sclerotiorum and host plants has been done, most of the work focused on the biochemical and molecular mechanism during S. sclerotiorum infection, screen of resistant variety, the clone of resistant gene and prevention and cure of plant disease caused by S. sclerotiorum. However, little work has been done on the effect of S. sclerotiorum infection on the photosynthetic apparatus of the host plants. The aim of the present study was to explore the effects of S. sclerotiorum infection on the photosynthetic performance and by which mechanism S. sclerotiorum affects the photosynthetic apparatus in host plants.In this study, tobacco plant was used as experimental martial.We firstly confirmed that the damage to photosynthetic apparatus during S. sclerotiorum infection was due to the effect of C2O42-. Then through measuring the photosynthetic gas exchange, oxygen evolution, chlorophyll a fluorescence transient, modulated light reflection of820nm (MR), H2O2accumulation and the activity of antioxidase in tobacco leaves treated with K2C2O4, we emphaticly studied the effect of C2O42-on photosynthetic electron transport, carbon assimilation and photoinhibition in tobacco leaves, analyzed the cause of photoinhibition aggravated by C2O42-, and discussed the possible relationship between photoinhibition and S. sclerotiorum infection.The main results are listed as below:(1) S. sclerotiorum infection decreased oxygen evolution rate and the maximum photochemical efficiency (Fv/Fm) in tobacco leaves, which indicates that S. sclerotiorum infection severely damaged photosynthetic apparatus. (2) The effect of H2C2O4(40mM, pH4.0), H3PO4(pH4.0) and HC1(pH4.0) on the chlorophyll a fluorescence transient were analyzed. The results showed that, with the same pH, H2CC2O4(40mM, pH4.0) damaged photosynthetic apparatus more severely than H3PO4and HC1did. Moreover, the damage extent of K2C2O4to photosynthetic apparatus was about70%of that caused by H2C2O4. The results suggest that C2O42-is the main reason for the enhanced photoinhibition and the damage to photosynthetic apparatus.(3) The effect of different concentration of K2C2O4solution (20,40,60mM) on photoinhibition was studied. Because120mM KC1had the same concentration of K+as60mM K2C2O4and with lower osmotic potential than60mM K2C2O4,120mM KC1was taken as the second control to preclude the effect of K+on the results. The results showed that K2C2O4treatment decreased Fv/Fm, the exciton efficiency of electron transport beyond Qa-(Ψo) and density of active reaction centers (RC/CSm) in tobacco leaves under high light and the decreasing extent amplified with the increase of K2C2O4concentration, but had no effect on Wk. It suggests that C2O42-mainly damaged the photosystem II (PSII) reaction center and the acceptor side but not the donor side of PSII, which was further supported by the fact that non-QA and non-QB reducing reaction center were both increased in K2C2O4treated leaves.(4) After treated under high light (800μmol m-2s-1) for2h, the tobacco leaves were treated with H2O (control), KC1and K2C2O4respectively and were placed under low light (50μmol m-2s-1) for recovery, the result of which showed that the PSII activity of K2C2O4treated leaves could not recover from photoinhibition. When the synthesis of D1protein was inhibited by a specific inhibitor (Chloramphenicol, CM), K2C2O4treatement did not enhance the damage to PSII any more. It was demonstrated that C2O42-mainly inhibited the repair of PSII through inhibiting the D1protein turnover.(5) It has been reported that the reactive oxygen species (ROS) is the key reason for the inhibition of D1protein turnover and enhancing of PSII photoinhibition. We observed an increase of ROS in K2C2O4treated leaves through histochemical detection, the results showed an oxidation stress in K2C2O4treated leaves. Therefore, the K2C2O4induced over production of ROS in tobacco leaves may be the direct reason for the D1protein inhibition and PSII photoinhibition in S. sclerotiorum infected leaves. And we also suggested that the decreased activity of antioxidases may be one of the reasons for the increased ROS in K2C2O4treated tobacco leaves.(6) We observed that the K2C2O4treatment decreased both the initial slope and the maximum value of Pn-CO2curve, which suggests an inhibition of carboxylation efficiency and ribulose1,5-biphosphate (RuBP) regeneration in K2C2O4tobacco leaves. The reduction of soluble sugar and starch in tobacco leaves further supported the conclusion.(7) When an inhibitor of Calvin cycle (Iodoacetamide, IAM) and K2C2O4were used to co-treated tobacco leaves, the further damage of PSII caused by K2C2O4was eliminated. It suggests that the enhancement of PSII photoinhibition caused by K2C2O4was through inhibiting Calvin cycle.(8) Through820nm detection technique, we demonstrated that the PSI activiy in tobacco leaves was not affected and the cyclic electron flow around PSI was decreased in K2C2O4treated leaves. The decrease in cyclic electron flow may be one of the reasons for the increase in ROS and PSII photoinhibition in K2C2O4treated leaves.(9) It has been reported that pathogen infection would lead to the chlorotic of the leaves. To explore if the pigment degradation of chlorophyll contributed to the decrease of photosynthetic performance, the pigment contents of the treated leaves were measured. The results showed that there were no difference in the chlorophyll a, b and carotinoid content between the leaves of different treatments. We speculate that the duration of the treatment was too short (only several hours) for the pigment to degradate in K2C2O4treated tobacco leaves. It suggests that the K2C2O4induced damage of photosynthetic apparatus and photosynthetic activity under high light in short time was not depended on the degradation of chlorophyll.(10) In the dark for long time (>10h), we observed a decrease in Fv/Fm, RC/CS and Ψ0in tobacco leaves treated with K2C2O4. The results suggest that K2C2O4could damage photosynthetic apparatus in the dark for long time, though no significant effect for short time, which may be related to the programmed cell death (PCD) induced by C2O42".In summary, we demonstrated that the enhancement of photoinhibition and damage of photosynthetic apparatus in the leaves infected by S. sclerotiorum was maily caused by C2O42-. By over production of ROS through inhibiting the carboxylation and RuBP regeneration, C2O422-inhibited the D1protein turnover and aggravated photoinhibition in tobacco leaves.