BACKGROUND
sclerotinia sclerotiorum  de bary is a necrotrophic fungal pathogen which causes disease in a wide range of plants, leading to enormous crop reduction  <cit> .
previous studies demonstrated that h2c2o <dig> is an important pathogenic determinant of s. sclerotiorum.
s. sclerotiorum mutants deficient in oxalate biosynthesis were shown to be less pathogenic than wild-type fungus, and enhancement of h2c2o <dig> degradation capacity was shown to enhance plant resistance to s. sclerotiorum  <cit> .
a great deal of researches have been conducted on the pathogenic mechanisms of s. sclerotiorum.
it was reported that s. sclerotiorum can maintain maximal activity of cell wall-degrading enzymes such as polygalacturonase through the acid environment provided by h2c2o <dig>  <cit> .
the cell wall is a natural barrier shield which protects plant tissues from pathogenic bacteria.
s. sclerotiorum can weaken the cell wall through chelation of ca2+ ions present in the cell wall with oxalic ions, breaking down ca2+-dependent signal transduction pathways in the host plant  <cit> .
nadph oxidase, which is involved in reactive oxygen species generation, is required for the pathogenic development and is important for ros regulation in the successful pathogenesis of s. sclerotiorum  <cit> .
recently, it was also reported that s. sclerotiorum  generates reducing conditions that suppress host defense responses, including the oxidative burst and callose deposition, during the initial stages of infection.
however, once infection is established, s. sclerotiorum benefits to its infection by inducing the generation of plant ros and programmed cell death   <cit> .
the leaves of host plant, the primary sites of photosynthesis, are the main targets of many pathogens.
the infection of pathogens to the leaves directly reduces photosynthetic performance, leading to drastic losses in crop yield.
the repression of photosynthesis in host plants induced by pathogens has been reported in many plant species.
for example, photosynthesis was decreased in barley infected by b. graminis  <cit> .
and down-regulation of photosystem ii quantum yield in host plants occurred during the infection of pseudomonas syringae  <cit> , albugo candida  <cit> , puccinia coronata and blumeria graminis  <cit> , as well as botrytis cinerea  <cit> .
it was also reported that the expression of sugar-regulated photosynthetic genes, such as the small subunit of ribulose- <dig> -bisphosphate carboxylase and chlorophyll a, b binding protein, in most cases, were down-regulated after pathogen infection  <cit> .
s. sclerotiorum infection could also decrease photosynthetic performance in host plants.
during s. sclerotiorum infection, h2c2o <dig> promotes the accumulation of osmotically active molecules, inducing stomatal opening and inhibiting aba induced stomatal closure, leading to foliar wilting  <cit> .
in our previous work, s. sclerotiorum infection was shown to induce the over-accumulation of h2o <dig> in cucumber leaves due to inhibition of the activity of catalase, damaging the functions of photosystem i  and photosystem ii   <cit> .
although h2c2o <dig> was determined to be the main toxin secreted by s. sclerotiorum, it is still unknown whether the destructive effect of s. sclerotiorum on the host photosynthetic apparatus is due to the acidity of h2c2o <dig> or the effect of the c2o42− anion, and if either of them has a deleterious effect, what is the mechanism of impairment of photosynthetic performance?
in order to address this question, we compared the effect of h2c2o <dig> acidity and c2o42− ion on the photosynthetic performance of tobacco leaves, and studied the effects of c2o42− on the photosynthetic electron transport chain, carbon assimilation, the production of ros and the synthesis of d <dig> protein in tobacco leaves under high-light treatment.
the psi activity and cyclic electron transport activity were also measured using modulated 820 nm reflection  techniques  <cit> .
RESULTS
effect of s. sclerotiorum infection on o <dig> evolution and psii activity of leaves
the o <dig> evolution rate reflects the capacity of the photosynthetic apparatus, including both the electron transport chain and carbon assimilation.
the fluorescence parameter fv/fm, providing an estimate of the maximum quantum yield of primary photochemistry, is widely used to reflect the extent of photoinhibition  <cit> .
both the photosynthetic o <dig> evolution rate and fv/fm decreased significantly in leaves infected with s. sclerotiorum when compared with controls, and the decreasing extent increased with the lasting of infection .
this result indicates that s. sclerotiorum infection significantly inhibited photosynthesis and aggravated the photoinhibition in tobacco leaves under high-light.figure  <dig>
changes in the photosynthetic o
2
evolution rate  and the maximum quantum yield of primary photochemistry  in tobacco leaves infected with
s. sclerotiorum
.
●, ○ indicate control and infected leaves, respectively.
control leaves were leaves without infection by s. sclerotiorum.
leaf discs used for o <dig> evolution rate determination and fv/fm measurement were cut within the region  <dig> cm away from the centre of the necrotic spot with a 10-mm cork borer.
different letters indicate significant differences between the leaves with different treatments .
values were means ± se .
the effect of h2c2o <dig> and other acids on the activity of psii
it has been proved that h2c2o <dig> is the primary pathogenic material secreted by s. sclerotiorum  <cit> .
additionally, injection of exogenous h2c2o <dig> can mimic disease symptoms of an actual fungal infection  <cit> .
so h2c2o <dig> was used in the following experiments to study the effect of s. sclerotiorum on the photosynthetic performance in host plants.
because the ph and concentration of h2c2o <dig> in the culture solution of s. sclerotiorum were  <dig>  and 40 mm respectively after 14 days growing of the s. sclerotiorum, 40 mm h2c2o <dig> with ph  <dig>   was used to treat leaves in this experiment.
in order to distinguish the effect of h2c2o4-mediated acidity from the effect of c2o42− anion on photosynthetic performance, the effects of acidity  provided by h2c2o <dig> , h3po <dig> and hcl, respectively, were compared to 40 mm k2c2o <dig>
the fv/fm decreased significantly in leaves treated with hcl , h3po <dig> , h2c2o <dig>  and k2c2o <dig>  after high-light treatment .
the decrease of fv/fm in hcl and h3po <dig> treated leaves was only slightly lower than that in ck leaves after high-light treatment.
however, h2c2o <dig> treatment significantly decreased fv/fm in tobacco leaves .
when k2c2o <dig> was used to eliminate h2c2o <dig> acidity in the experiment, the psii activity was also severely inhibited under high-light.
the extent of k2c2o <dig> inhibition of fv/fm reached  <dig> % of the inhibition induced by h2c2o <dig> .
meanwhile, the fm of the normalized fluorescence transient in k2c2o <dig> treated leaves decreased much more than that in hcl and h3po <dig> treated leaves , and the decreased extent was only a bit smaller than that in h2c2o <dig> treated leaves.
the results indicate that the influence of h2c2o <dig> on photosynthetic performance was mainly caused by the c2o42− anion.figure  <dig>
effect of hcl , h
3
po
4
, h
2
c
2
o
4
, k
2
c
2
o
4
and kcl  treatment on f
v
/f
m
and ojip curves  in tobacco leaves.
■ indicates leaves before treatment; □ indicates leaves 2 hours after high-light treatment.
leaf discs  were infiltrated with hcl , h3po <dig> , h2c2o <dig> , k2c2o <dig>  and kcl  under darkness for 3 h, followed by exposure to high-light  for 2 hours.
ck were leaves without any reagent treatment.
different letters indicate significant differences between leaves with different treatments .
values were means ± se .
the effect of different concentrations of k2c2o <dig> on the activity of psii
in leaves treated with  <dig>   <dig>  60 mm k2c2o <dig> and 120 mm kcl, fv/fm decreased significantly in high light .
when the treated leaves were placed in low light  for recovery after photoinhibition treatment, the fv/fm in all leaves recovered to a large extent.
however, the fv/fm in k2c2o <dig> treated leaves recovered less than that in the control and kcl treated leaves .
nonetheless, no significant differences were observed in fv/fm between different treatment groups in the dark .
there was no significant difference in fv/fm between kcl treated leaves and the control leaves either.
because 120 mm kcl has the same concentration of k+ as 60 mm k2c2o <dig> does, and it has lower osmotic potential than 60 mm k2c2o <dig>  the effect of k+ and osmotic stress on the result was then neglected.figure  <dig>
effect of different concentrations of k
2
c
2
o
4
and kcl on f
v
/f
m
and ojip curves  in tobacco leaves.
in a, leaves were kept in the dark from 0– <dig> h,  <dig> μmol m− <dig> s− <dig> light from 3– <dig> h, and  <dig> μmol · m− <dig> · s− <dig> light from 5– <dig> h to recover.
in b, the leaves were kept in the dark the duration of the study.
ck were leaves without any reagent treatment.
different letters indicate significant differences between leaves with different treatments .
values were means ± se .
in high light  for 2 hours, the electron transport rate  and photochemical quenching  in k2c2o <dig> treated leaves decreased much more than that in control and kcl treated leaves, meanwhile, npq was higher in k2c2o <dig> treated leaves than that in the leaves with other treatment .
this result further indicates that k2c2o <dig> treatment aggravates psii photoinhibition in tobacco leaves under high-light.figure  <dig>
effect of k
2
c
2
o
4
and kcl  treatment on etr , npq  and qp  in tobacco leaves.
leaf discs  were infiltrated with k2c2o <dig>  and kcl  under darkness for 3 h, followed by exposure to intense light  for 2 hours.
ck were leaves without any reagent treatment.
different letters indicate significant differences between leaves with different treatments .
values were means ± se .
in the presence of chloramphenicol , an inhibitor of de novo d <dig> protein synthesis  <cit> , k2c2o <dig> treatment did not aggravate the decrease of fv/fm .
and the large difference in p point of the ojip curves between different treatments was eliminated by cm treatment, which indicates that the inhibition of d <dig> protein by c2o42− accelerated the photoinhibition of psii in leaves treated with h2c2o <dig> under high-light.figure  <dig>
the effect of  <dig> mm k
2
c
2
o
4
and  <dig> mm kcl treatment on the f
v
/f
m
and ojip curves  in tobacco leaves in the presence of chloramphenicol .
■, − cm; □, + cm.
leaf discs  were immersed into solutions with 1 mm cm for 3 h in the dark, then kept in high light  for 2 h. fv/fm and Ψo were measured after 2 h high light treatment.
ck were leaves without k2c2o <dig> and kcl treatment.
different letters indicate significant differences between leaves with different treatments .
values were means ± se .
the effect of k2c2o <dig> treatment on the accumulation of hydrogen peroxide
as shown in figure  <dig>  k2c2o <dig> treatment obviously enhanced the accumulation of h2o <dig> in tobacco leaves under high light when compared with control and kcl treated leaves, which indicates that k2c2o <dig> treated leaves suffered from greater photo-oxidative stress.figure  <dig>
the accumulation of h
2
o
2
in tobacco leaves after treatment with h
2
o, 80 mm kcl and 40 mm k
2
c
2
o
4
under high light  for 2 hours.
histochemical detection of h2o <dig> production with 1 mg/ml  <dig>  3-diaminobenzidine  staining.
ck were leaves without k2c2o <dig> and kcl treatment.
representative images from five independent experiments are shown in the figure.
the effect of k2c2o <dig> on carbon assimilation
a photosynthetic model suggests that co <dig> assimilation in c <dig> plants is limited by the rate of rubp regeneration at high levels of co <dig>  and is limited by the efficiency of rubisco at lower levels of co <dig>  <cit> .
in tobacco leaves treated with k2c2o <dig>  carboxylation efficiency  and net photosynthetic rate  at saturated co <dig>  both decreased severely compared to the control, at magnitudes which increased with k2c2o <dig> concentration .
however, no significant decreases in ce and am were observed in leaves treated with 120 mm kcl.
it suggests that c2o42− inhibited both rubisco activity and rubp regeneration.
in addition, after exposed to light for 2 h, the contents of both soluble sugar and starch in k2c2o <dig> treated leaves were significantly lower than those in control and kcl treated leaves , which demonstrates that c2o42− significantly inhibited calvin cycle.figure  <dig>
relative photosynthetic rates at saturated co
2
, and carboxylation efficiency  of tobacco leaves treated with different concentrations  of k
2
c
2
o
4
and  <dig> mm kcl.
the petioles of detached leaves were dipped into treatment solutions before measurement in the dark for 3 hours.
ck were leaves without k2c2o <dig> and kcl treatment.
different letters indicate significant differences between leaves with different treatments .
values shown are means ± se .
the effect of k
2
c
2
o
4
and kcl treatment on the content of soluble sugar  and starch  in tobacco leaves under light  for  <dig> hours.
the “dark” were dark adapted leaves without any reagent treatment.
different letters indicate significant differences between leaves with different treatments .
values shown are means ± se .
to further investigate the effect of k2c2o <dig> on carbon assimilation, leaves were pretreated with iodoacetamide , an inhibitor of the calvin cycle  <cit> .
in the presence of iam, no significant differences were observed in fv/fm between k2c2o <dig> treated leaves and control leaves after high light treatment .
this result implies that when co <dig> assimilation was inhibited, k2c2o <dig> didn’t further aggravate the photoinhibition under high light proving that the severe photoinhibition caused by the k2c2o <dig> may be due in part to inhibition of the calvin cycle.
the inference was also supported by the fact that iam treatment eliminated the difference in ojip curves between k2c2o <dig> treated leaves and ck.figure  <dig>
the effect of  <dig> mm k
2
c
2
o
4
and  <dig> mm kcl treatment on the f
v
/f
m
and ojip curves  in tobacco leaves under light  with or without  <dig> mm iodoacetamide .
ck were leaves without k2c2o <dig> and kcl treatment.
different letters indicate significant differences between leaves with different treatments .
values shown are means ± se .
the effect of k2c2o <dig> on psi activity and cyclic electron flow
the psi activities in control leaves, and leaves treated with kcl and k2c2o <dig> were measured.
no significant differences were observed in psi activity between leaves with different treatments .
however, the initial increase rate of mr820nm in k2c2o <dig> treated leaves after far-red illumination significantly decreased , which indicates a decrease in cyclic electron flow in k2c2o <dig> treated leaves.figure  <dig>
the effect of k
2
c
2
o
4
and kcl treatment on the psi activity.
the psi activity of the treated leaves were measured with an m-pea .
the induction curve of mr <dig> nm of the leaves obtained by saturating red light showed a fast oxidation phase and a following reduction phase.
the initial slope of the oxidation phase of mr820nm at the beginning of the saturated red light indicates the capability of p <dig> to get oxidized, which is used to reflect the activity of psi.
the effect of k
2
c
2
o
4
and kcl treatment on the cyclic electron flow.
the treated leaves were illuminated with a 20 s far-red light, then turn off the light and record the changes of the mr820nm signal.
the initial rate of mr820nm indicates the activity of cyclic electron flow.
discussion
s. sclerotiorum infection significantly inhibited photosynthesis , which suggests that the photosynthetic apparatus is a major pathogenic target.
in this study, we demonstrated that h2c2o <dig>  the most important pathogenic determinant of s. sclerotiorum infection, inhibited photosynthetic activity in tobacco leaves more severely than hcl and h3po <dig> did at the same ph.
the c2o42− anion appeared to play a more important role in damaging the photosynthetic performance than the acidity did, which was supported by the fact that the inhibition of k2c2o <dig> on psii activity reached about 70% of the effect observed with h2c2o <dig> .
therefore, k2c2o <dig> was used to study the mechanisms by which s. sclerotiorum affects photosynthetic performance in tobacco leaves.
it has been known that the infection of s. sclerotiorum decreases chlorophyll content and causes the appearance of chlorotic symptom in host plants.
however, the decrease in chlorophyll content often occurs after infection for several days  <cit> , in our experiment, the treatment period is only about 5 h, so the decrease in chlorophyll content didn’t occur in the experiment .
the result indicates that the effect of k2c2o <dig> on photosynthetic apparatus does not depend on the degradation of chlorophyll.
fv/fm, etr and qp decreased significantly in k2c2o <dig> treated leaves but the npq increased, indicating that the activity of psii was significantly damaged by c2o42−.we propose that during s. sclerotiorum infection, the decreased psii activity was probably due to the damage to the reaction centre and acceptor side of psii by c2o42− secreted by s. sclerotiorum because there was no difference observed in wk  between k2c2o <dig> treated leaves and control leaves , and Ψo  decreased in k2c2o <dig> treated leaves .
in the dark, no significant difference was observed in fv/fm between k2c2o <dig> treated leaves and control , demonstrating that c2o42− inhibited the photosynthetic electron transfer chain in an indirect, light-dependent manner.
psii photoinhibition occurs when the absorbed light is more than it can be used by photochemistry  <cit> .
however, photosynthetic organisms are able to overcome psii photoinhibition by rapidly and efficiently repairing the damage, which requires the synthesis of proteins de novo, such as d <dig>  <cit> .
under high-light, the psii activity depends on the balance between the rates of photodamage and repair; consequently, photoinhibition of psii becomes apparent when the rate of photodamage exceeds the rate of repair  <cit> .
as observed in the study, in the presence of cm, the k2c2o4-mediated decrease of fv/fm and changes of ojip curves were eliminated , which indicates that inactivation of psii by the c2o42− was largely caused by inhibition of d <dig> protein synthesis.
recently, it was suggested that all environmental stresses enhance photoinhibition through an indirect method - promoting the production of ros to inhibit the repair of the d <dig> protein  <cit> .
in this study, a marked increase of h2o <dig> was observed in leaves treated with c2o42− , inferring that the increase of the ros may inhibit d <dig> protein synthesis.
however, further study is needed to clarify whether d <dig> protein synthesis was inhibited by accumulation of ros caused by c2o42−, or via some combination of the accumulation of ros and c2o42− direct effect.
the chloroplast photosynthetic electron transport chain is one of the major sites of ros production in leaves of green plants  <cit> .
the photosynthetic electron transport and carbon assimilation work coordinately under normal physiological conditions.
the damage or inhibition of either one of the two components would lead to increased ros production.
the fact that both the ce and am were significantly inhibited by c2o42− indicates that carbon assimilation processes, including both rubisco activity and rubp regeneration , were inhibited by c2o42− under high-light, which is further supported by the decrease in the content of soluble sugar and starch in k2c2o <dig> treated leaves .
we suggest that the over accumulation of ros resulted from the inhibition of co <dig> assimilation enhanced c2o42− mediated photoinhibition.
this was further supported by the fact that iam treatment eliminated the decrease in fv/fm  and the changes of ojip curves induced by k2c2o <dig> under high-light.
it is known that c2o42− can react with many metal ions to form chelate compounds.
rubisco and fructose- <dig>  6-bisphosphatase are key enzymes in calvin cycle.
it has been reported that ca2+ and mg2+ play important roles in the regulation of the activity of chloroplast fructose- <dig> -bisphosphatase  <cit> , and mg2+ has a enhancing effect on the rubisco activity  <cit> .
we presume the increased c2o42− in plant cells may decrease the activity of rubisco or fructose- <dig> -bisphosphatase through forming chelate compounds with ca2+ or mg2+ in chloroplast.
moreover, the signal role of h2c2o <dig> during the infection of s. sclerotiorum has been demonstrated  <cit> , the increased c2o42− in plant cells may also interfere with co <dig> assimilation through signal transduction.
however, to determine the specific mechanism by which c2o42− inhibit the calvin cycle needs further studies.
under high-light treatment, though psi activity was not damaged by c2o42− , cyclic electron flow around psi was decreased by k2c2o <dig> treatment .
the over-reducing of psi acceptors, always leading to the increase of the generation of superoxide, would be prevented by an increase of cyclic electron flow around psi  <cit> .
moreover, the cyclic electron flow can also decrease the probability that singlet oxygen generates within psii through charge recombination  <cit> .
further studies are needed to clarify whether the decrease in cyclic electron flow induced by c2o42− is correlated to the increase in ros generation and the enhancement of psii photoinhibition in k2c2o <dig> treated leaves.
our experiment demonstrated that it is the c2o42− ion secreted by s. sclerotiorum rather than the decrease in ph caused by the h2c2o <dig> that mainly induces the damage to photosynthetic apparatus.
however, we did not exclude that necrosis may also cause inhibition of calvin cycle and psii activity.
since the c2o42− ion plays a more important role in impairing photosynthetic apparatus than acidity does, it reasonable to infer that the inhibition of calvin cycle and psii activity by necrosis in s. sclerotiorum infected leaves is mainly induced by the c2o42− ion secreted by s. sclerotiorum.
fo is the fluorescence emitted by antenna pigment of psii with open reaction center.
chlorophyll content and fo all showed no significant difference among the leaves treated with k2c2o <dig>  ck and kcl, which indicates that the antenna of psii might not affected by c2o42− in our experiment .
CONCLUSIONS
this study demonstrated that h2c2o <dig> secreted by s. sclerotiorum enhanced photoinhibition, mainly by the effect of the c2o42− ion.
c2o42− led to the decrease of both the activity of rubisco and rubp regeneration, leading to the accumulation of h2o <dig> in the chloroplast.
the over accumulation of h2o <dig> inhibited the turnover of the d <dig> protein.
this is likely the primary mechanism by which s. sclerotiorum infection affects the photosynthetic performance of tobacco leaves.
further studies are needed to explore whether c2o42− has a direct effect on d <dig> protein synthesis, and to elucidate the detailed mechanisms by which c2o42− inhibits the activity of rubisco and rubp regeneration in tobacco leaves.
and if the inhibition of rubisco activity and rubp regeneration is mediated by the signal effect of c2o42− and if the decrease in psii activity caused by c2o42− is involved in the pcd induced by s.sclerotiorum remain to be elucidated in future work.

 sclerotinia sclerotiorum de bary is a necrotrophic fungal pathogen which causes disease in a wide range of plants, leading to enormous crop reduction. previous studies demonstrated that h2c2o is an important pathogenic determinant of s. sclerotiorum. s. sclerotiorum mutants deficient in oxalate biosynthesis were shown to be less pathogenic than wild-type fungus, and enhancement of h2c2o degradation capacity was shown to enhance plant resistance to s. sclerotiorum. a great deal of researches have been conducted on the pathogenic mechanisms of s. sclerotiorum. it was reported that s. sclerotiorum can maintain maximal activity of cell wall-degrading enzymes such as polygalacturonase through the acid environment provided by h2c2o. the cell wall is a natural barrier shield which protects plant tissues from pathogenic bacteria. s. sclerotiorum can weaken the cell wall through chelation of ca2+ ions present in the cell wall with oxalic ions, breaking down ca2+-dependent signal transduction pathways in the host plant. nadph oxidase, which is involved in reactive oxygen species generation, is required for the pathogenic development and is important for ros regulation in the successful pathogenesis of s. sclerotiorum. recently, it was also reported that s. sclerotiorum generates reducing conditions that suppress host defense responses, including the oxidative burst and callose deposition, during the initial stages of infection. however, once infection is established, s. sclerotiorum benefits to its infection by inducing the generation of plant ros and programmed cell death. the leaves of host plant, the primary sites of photosynthesis, are the main targets of many pathogens. the infection of pathogens to the leaves directly reduces photosynthetic performance, leading to drastic losses in crop yield. the repression of photosynthesis in host plants induced by pathogens has been reported in many plant species. for example, photosynthesis was decreased in barley infected by b. graminis. and down-regulation of photosystem ii quantum yield in host plants occurred during the infection of pseudomonas syringae, albugo candida, puccinia coronata and blumeria graminis, as well as botrytis cinerea. it was also reported that the expression of sugar-regulated photosynthetic genes, such as the small subunit of ribulose- -bisphosphate carboxylase and chlorophyll a, b binding protein, in most cases, were down-regulated after pathogen infection. s. sclerotiorum infection could also decrease photosynthetic performance in host plants. during s. sclerotiorum infection, h2c2o promotes the accumulation of osmotically active molecules, inducing stomatal opening and inhibiting aba induced stomatal closure, leading to foliar wilting. in our previous work, s. sclerotiorum infection was shown to induce the over-accumulation of h2o in cucumber leaves due to inhibition of the activity of catalase, damaging the functions of photosystem i and photosystem ii. although h2c2o was determined to be the main toxin secreted by s. sclerotiorum, it is still unknown whether the destructive effect of s. sclerotiorum on the host photosynthetic apparatus is due to the acidity of h2c2o or the effect of the c2o42− anion, and if either of them has a deleterious effect, what is the mechanism of impairment of photosynthetic performance? in order to address this question, we compared the effect of h2c2o acidity and c2o42− ion on the photosynthetic performance of tobacco leaves, and studied the effects of c2o42− on the photosynthetic electron transport chain, carbon assimilation, the production of ros and the synthesis of d protein in tobacco leaves under high-light treatment. the psi activity and cyclic electron transport activity were also measured using modulated 820 nm reflection techniques.  effect of s. sclerotiorum infection on o evolution and psii activity of leaves the o evolution rate reflects the capacity of the photosynthetic apparatus, including both the electron transport chain and carbon assimilation. the fluorescence parameter fv/fm, providing an estimate of the maximum quantum yield of primary photochemistry, is widely used to reflect the extent of photoinhibition. both the photosynthetic o evolution rate and fv/fm decreased significantly in leaves infected with s. sclerotiorum when compared with controls, and the decreasing extent increased with the lasting of infection. this result indicates that s. sclerotiorum infection significantly inhibited photosynthesis and aggravated the photoinhibition in tobacco leaves under high-light.figure changes in the photosynthetic o 2 evolution rate and the maximum quantum yield of primary photochemistry in tobacco leaves infected with s. sclerotiorum. ●, ○ indicate control and infected leaves, respectively. control leaves were leaves without infection by s. sclerotiorum. leaf discs used for o evolution rate determination and fv/fm measurement were cut within the region cm away from the centre of the necrotic spot with a 10-mm cork borer. different letters indicate significant differences between the leaves with different treatments. values were means ± se. the effect of h2c2o and other acids on the activity of psii it has been proved that h2c2o is the primary pathogenic material secreted by s. sclerotiorum. additionally, injection of exogenous h2c2o can mimic disease symptoms of an actual fungal infection. so h2c2o was used in the following experiments to study the effect of s. sclerotiorum on the photosynthetic performance in host plants. because the ph and concentration of h2c2o in the culture solution of s. sclerotiorum were and 40 mm respectively after 14 days growing of the s. sclerotiorum, 40 mm h2c2o with ph  was used to treat leaves in this experiment. in order to distinguish the effect of h2c2o4-mediated acidity from the effect of c2o42− anion on photosynthetic performance, the effects of acidity provided by h2c2o, h3po and hcl, respectively, were compared to 40 mm k2c2o the fv/fm decreased significantly in leaves treated with hcl, h3po, h2c2o and k2c2o after high-light treatment. the decrease of fv/fm in hcl and h3po treated leaves was only slightly lower than that in ck leaves after high-light treatment. however, h2c2o treatment significantly decreased fv/fm in tobacco leaves. when k2c2o was used to eliminate h2c2o acidity in the experiment, the psii activity was also severely inhibited under high-light. the extent of k2c2o inhibition of fv/fm reached % of the inhibition induced by h2c2o. meanwhile, the fm of the normalized fluorescence transient in k2c2o treated leaves decreased much more than that in hcl and h3po treated leaves, and the decreased extent was only a bit smaller than that in h2c2o treated leaves. the results indicate that the influence of h2c2o on photosynthetic performance was mainly caused by the c2o42− anion.figure effect of hcl, h 3 po 4, h 2 c 2 o 4, k 2 c 2 o 4 and kcl treatment on f v /f m and ojip curves in tobacco leaves. ■ indicates leaves before treatment; □ indicates leaves 2 hours after high-light treatment. leaf discs were infiltrated with hcl, h3po, h2c2o, k2c2o and kcl under darkness for 3 h, followed by exposure to high-light for 2 hours. ck were leaves without any reagent treatment. different letters indicate significant differences between leaves with different treatments. values were means ± se. the effect of different concentrations of k2c2o on the activity of psii in leaves treated with 60 mm k2c2o and 120 mm kcl, fv/fm decreased significantly in high light. when the treated leaves were placed in low light for recovery after photoinhibition treatment, the fv/fm in all leaves recovered to a large extent. however, the fv/fm in k2c2o treated leaves recovered less than that in the control and kcl treated leaves. nonetheless, no significant differences were observed in fv/fm between different treatment groups in the dark. there was no significant difference in fv/fm between kcl treated leaves and the control leaves either. because 120 mm kcl has the same concentration of k+ as 60 mm k2c2o does, and it has lower osmotic potential than 60 mm k2c2o the effect of k+ and osmotic stress on the result was then neglected.figure effect of different concentrations of k 2 c 2 o 4 and kcl on f v /f m and ojip curves in tobacco leaves. in a, leaves were kept in the dark from 0– h, μmol m− s− light from 3– h, and μmol · m− · s− light from 5– h to recover. in b, the leaves were kept in the dark the duration of the study. ck were leaves without any reagent treatment. different letters indicate significant differences between leaves with different treatments. values were means ± se. in high light for 2 hours, the electron transport rate and photochemical quenching in k2c2o treated leaves decreased much more than that in control and kcl treated leaves, meanwhile, npq was higher in k2c2o treated leaves than that in the leaves with other treatment. this result further indicates that k2c2o treatment aggravates psii photoinhibition in tobacco leaves under high-light.figure effect of k 2 c 2 o 4 and kcl treatment on etr, npq and qp in tobacco leaves. leaf discs were infiltrated with k2c2o and kcl under darkness for 3 h, followed by exposure to intense light for 2 hours. ck were leaves without any reagent treatment. different letters indicate significant differences between leaves with different treatments. values were means ± se. in the presence of chloramphenicol, an inhibitor of de novo d protein synthesis, k2c2o treatment did not aggravate the decrease of fv/fm. and the large difference in p point of the ojip curves between different treatments was eliminated by cm treatment, which indicates that the inhibition of d protein by c2o42− accelerated the photoinhibition of psii in leaves treated with h2c2o under high-light.figure the effect of mm k 2 c 2 o 4 and mm kcl treatment on the f v /f m and ojip curves in tobacco leaves in the presence of chloramphenicol. ■, − cm; □, + cm. leaf discs were immersed into solutions with 1 mm cm for 3 h in the dark, then kept in high light for 2 h. fv/fm and Ψo were measured after 2 h high light treatment. ck were leaves without k2c2o and kcl treatment. different letters indicate significant differences between leaves with different treatments. values were means ± se. the effect of k2c2o treatment on the accumulation of hydrogen peroxide as shown in figure  k2c2o treatment obviously enhanced the accumulation of h2o in tobacco leaves under high light when compared with control and kcl treated leaves, which indicates that k2c2o treated leaves suffered from greater photo-oxidative stress.figure the accumulation of h 2 o 2 in tobacco leaves after treatment with h 2 o, 80 mm kcl and 40 mm k 2 c 2 o 4 under high light for 2 hours. histochemical detection of h2o production with 1 mg/ml 3-diaminobenzidine staining. ck were leaves without k2c2o and kcl treatment. representative images from five independent experiments are shown in the figure. the effect of k2c2o on carbon assimilation a photosynthetic model suggests that co assimilation in c plants is limited by the rate of rubp regeneration at high levels of co and is limited by the efficiency of rubisco at lower levels of co. in tobacco leaves treated with k2c2o carboxylation efficiency and net photosynthetic rate at saturated co both decreased severely compared to the control, at magnitudes which increased with k2c2o concentration. however, no significant decreases in ce and am were observed in leaves treated with 120 mm kcl. it suggests that c2o42− inhibited both rubisco activity and rubp regeneration. in addition, after exposed to light for 2 h, the contents of both soluble sugar and starch in k2c2o treated leaves were significantly lower than those in control and kcl treated leaves, which demonstrates that c2o42− significantly inhibited calvin cycle.figure relative photosynthetic rates at saturated co 2, and carboxylation efficiency of tobacco leaves treated with different concentrations of k 2 c 2 o 4 and mm kcl. the petioles of detached leaves were dipped into treatment solutions before measurement in the dark for 3 hours. ck were leaves without k2c2o and kcl treatment. different letters indicate significant differences between leaves with different treatments. values shown are means ± se. the effect of k 2 c 2 o 4 and kcl treatment on the content of soluble sugar and starch in tobacco leaves under light for hours. the “dark” were dark adapted leaves without any reagent treatment. different letters indicate significant differences between leaves with different treatments. values shown are means ± se. to further investigate the effect of k2c2o on carbon assimilation, leaves were pretreated with iodoacetamide, an inhibitor of the calvin cycle. in the presence of iam, no significant differences were observed in fv/fm between k2c2o treated leaves and control leaves after high light treatment. this result implies that when co assimilation was inhibited, k2c2o didn’t further aggravate the photoinhibition under high light proving that the severe photoinhibition caused by the k2c2o may be due in part to inhibition of the calvin cycle. the inference was also supported by the fact that iam treatment eliminated the difference in ojip curves between k2c2o treated leaves and ck.figure the effect of mm k 2 c 2 o 4 and mm kcl treatment on the f v /f m and ojip curves in tobacco leaves under light with or without mm iodoacetamide. ck were leaves without k2c2o and kcl treatment. different letters indicate significant differences between leaves with different treatments. values shown are means ± se. the effect of k2c2o on psi activity and cyclic electron flow the psi activities in control leaves, and leaves treated with kcl and k2c2o were measured. no significant differences were observed in psi activity between leaves with different treatments. however, the initial increase rate of mr820nm in k2c2o treated leaves after far-red illumination significantly decreased, which indicates a decrease in cyclic electron flow in k2c2o treated leaves.figure the effect of k 2 c 2 o 4 and kcl treatment on the psi activity. the psi activity of the treated leaves were measured with an m-pea. the induction curve of mr nm of the leaves obtained by saturating red light showed a fast oxidation phase and a following reduction phase. the initial slope of the oxidation phase of mr820nm at the beginning of the saturated red light indicates the capability of p to get oxidized, which is used to reflect the activity of psi. the effect of k 2 c 2 o 4 and kcl treatment on the cyclic electron flow. the treated leaves were illuminated with a 20 s far-red light, then turn off the light and record the changes of the mr820nm signal. the initial rate of mr820nm indicates the activity of cyclic electron flow. discussion s. sclerotiorum infection significantly inhibited photosynthesis, which suggests that the photosynthetic apparatus is a major pathogenic target. in this study, we demonstrated that h2c2o the most important pathogenic determinant of s. sclerotiorum infection, inhibited photosynthetic activity in tobacco leaves more severely than hcl and h3po did at the same ph. the c2o42− anion appeared to play a more important role in damaging the photosynthetic performance than the acidity did, which was supported by the fact that the inhibition of k2c2o on psii activity reached about 70% of the effect observed with h2c2o. therefore, k2c2o was used to study the mechanisms by which s. sclerotiorum affects photosynthetic performance in tobacco leaves. it has been known that the infection of s. sclerotiorum decreases chlorophyll content and causes the appearance of chlorotic symptom in host plants. however, the decrease in chlorophyll content often occurs after infection for several days, in our experiment, the treatment period is only about 5 h, so the decrease in chlorophyll content didn’t occur in the experiment. the result indicates that the effect of k2c2o on photosynthetic apparatus does not depend on the degradation of chlorophyll. fv/fm, etr and qp decreased significantly in k2c2o treated leaves but the npq increased, indicating that the activity of psii was significantly damaged by c2o42−.we propose that during s. sclerotiorum infection, the decreased psii activity was probably due to the damage to the reaction centre and acceptor side of psii by c2o42− secreted by s. sclerotiorum because there was no difference observed in wk between k2c2o treated leaves and control leaves, and Ψo decreased in k2c2o treated leaves. in the dark, no significant difference was observed in fv/fm between k2c2o treated leaves and control, demonstrating that c2o42− inhibited the photosynthetic electron transfer chain in an indirect, light-dependent manner. psii photoinhibition occurs when the absorbed light is more than it can be used by photochemistry. however, photosynthetic organisms are able to overcome psii photoinhibition by rapidly and efficiently repairing the damage, which requires the synthesis of proteins de novo, such as d. under high-light, the psii activity depends on the balance between the rates of photodamage and repair; consequently, photoinhibition of psii becomes apparent when the rate of photodamage exceeds the rate of repair. as observed in the study, in the presence of cm, the k2c2o4-mediated decrease of fv/fm and changes of ojip curves were eliminated, which indicates that inactivation of psii by the c2o42− was largely caused by inhibition of d protein synthesis. recently, it was suggested that all environmental stresses enhance photoinhibition through an indirect method - promoting the production of ros to inhibit the repair of the d protein. in this study, a marked increase of h2o was observed in leaves treated with c2o42−, inferring that the increase of the ros may inhibit d protein synthesis. however, further study is needed to clarify whether d protein synthesis was inhibited by accumulation of ros caused by c2o42−, or via some combination of the accumulation of ros and c2o42− direct effect. the chloroplast photosynthetic electron transport chain is one of the major sites of ros production in leaves of green plants. the photosynthetic electron transport and carbon assimilation work coordinately under normal physiological conditions. the damage or inhibition of either one of the two components would lead to increased ros production. the fact that both the ce and am were significantly inhibited by c2o42− indicates that carbon assimilation processes, including both rubisco activity and rubp regeneration, were inhibited by c2o42− under high-light, which is further supported by the decrease in the content of soluble sugar and starch in k2c2o treated leaves. we suggest that the over accumulation of ros resulted from the inhibition of co assimilation enhanced c2o42− mediated photoinhibition. this was further supported by the fact that iam treatment eliminated the decrease in fv/fm and the changes of ojip curves induced by k2c2o under high-light. it is known that c2o42− can react with many metal ions to form chelate compounds. rubisco and fructose- 6-bisphosphatase are key enzymes in calvin cycle. it has been reported that ca2+ and mg2+ play important roles in the regulation of the activity of chloroplast fructose- -bisphosphatase, and mg2+ has a enhancing effect on the rubisco activity. we presume the increased c2o42− in plant cells may decrease the activity of rubisco or fructose- -bisphosphatase through forming chelate compounds with ca2+ or mg2+ in chloroplast. moreover, the signal role of h2c2o during the infection of s. sclerotiorum has been demonstrated, the increased c2o42− in plant cells may also interfere with co assimilation through signal transduction. however, to determine the specific mechanism by which c2o42− inhibit the calvin cycle needs further studies. under high-light treatment, though psi activity was not damaged by c2o42−, cyclic electron flow around psi was decreased by k2c2o treatment. the over-reducing of psi acceptors, always leading to the increase of the generation of superoxide, would be prevented by an increase of cyclic electron flow around psi. moreover, the cyclic electron flow can also decrease the probability that singlet oxygen generates within psii through charge recombination. further studies are needed to clarify whether the decrease in cyclic electron flow induced by c2o42− is correlated to the increase in ros generation and the enhancement of psii photoinhibition in k2c2o treated leaves. our experiment demonstrated that it is the c2o42− ion secreted by s. sclerotiorum rather than the decrease in ph caused by the h2c2o that mainly induces the damage to photosynthetic apparatus. however, we did not exclude that necrosis may also cause inhibition of calvin cycle and psii activity. since the c2o42− ion plays a more important role in impairing photosynthetic apparatus than acidity does, it reasonable to infer that the inhibition of calvin cycle and psii activity by necrosis in s. sclerotiorum infected leaves is mainly induced by the c2o42− ion secreted by s. sclerotiorum. fo is the fluorescence emitted by antenna pigment of psii with open reaction center. chlorophyll content and fo all showed no significant difference among the leaves treated with k2c2o ck and kcl, which indicates that the antenna of psii might not affected by c2o42− in our experiment.  this study demonstrated that h2c2o secreted by s. sclerotiorum enhanced photoinhibition, mainly by the effect of the c2o42− ion. c2o42− led to the decrease of both the activity of rubisco and rubp regeneration, leading to the accumulation of h2o in the chloroplast. the over accumulation of h2o inhibited the turnover of the d protein. this is likely the primary mechanism by which s. sclerotiorum infection affects the photosynthetic performance of tobacco leaves. further studies are needed to explore whether c2o42− has a direct effect on d protein synthesis, and to elucidate the detailed mechanisms by which c2o42− inhibits the activity of rubisco and rubp regeneration in tobacco leaves. and if the inhibition of rubisco activity and rubp regeneration is mediated by the signal effect of c2o42− and if the decrease in psii activity caused by c2o42− is involved in the pcd induced by s.sclerotiorum remain to be elucidated in future work. 
this study demonstrated that the damage to the photosynthetic apparatus induced by s. sclerotiorum is not only caused by the acidity of h2c2o but also by c2o42− which plays a much more important role in damaging the photosynthetic apparatus. an observed decrease in photosynthetic performance is the primary reason for the reduction of crop yield induced by s. sclerotiorum. it was observed that k2c2o treatment inhibited the rate of rubp regeneration and carboxylation efficiency. c2o42− inhibits psii activity, as well as the rate of rubp regeneration and carboxylation efficiency, leading to the over production of reactive oxygen species. these results verify that the acidity of the h2c2o secreted by s. sclerotiorum was only partially responsible for the observed decreases in photosynthesis. the h2c2o is the main pathogenic material secreted by s. sclerotiorum, but the effects of h2c2o acidity and the c2o42− ion on photosynthetic performance remain unknown. s. sclerotiorum infection significantly decreased photosynthetic o evolution and the maximum quantum yield of photosystem ii in tobacco leaves under high-light. treatment with 40 mm k2c2o decreased fv/fm by about 70% of the levels observed under 40 mm h2c2o which further demonstrates that c2o42− was the primary factor that impaired photosynthetic performance during s. sclerotiorum infection. sclerotinia sclerotiorum de bary is a necrotrophic fungal pathogen which causes disease in a wide range of plants. in the presence of a carbon assimilation inhibitor, k2c2o treatment did not further decrease photosynthetic performance, which infers that c2o42− inhibited psii activity partly by repressing the carbon assimilation. in addition, it was showed that c2o42− treatment inhibited the psii activity but not the psi activity. h2c2o with ph  also significantly decreased photosynthetic performance. however, treatment with h3po and hcl at the same ph as h2c2o caused much less decrease in photosynthetic performance than h2c2o did. k2c2o treatment did not further decrease photosynthetic performance when d protein synthesis was fully inhibited, indicating that c2o42− inhibited psii by repressing d protein synthesis.
 sclerotinia sclerotiorum de bary is a necrotrophic fungal pathogen which causes disease in a wide range of plants. an observed decrease in photosynthetic performance is the primary reason for the reduction of crop yield induced by s. sclerotiorum. the h2c2o is the main pathogenic material secreted by s. sclerotiorum, but the effects of h2c2o acidity and the c2o42− ion on photosynthetic performance remain unknown.  s. sclerotiorum infection significantly decreased photosynthetic o evolution and the maximum quantum yield of photosystem ii in tobacco leaves under high-light. h2c2o with ph  also significantly decreased photosynthetic performance. however, treatment with h3po and hcl at the same ph as h2c2o caused much less decrease in photosynthetic performance than h2c2o did. these results verify that the acidity of the h2c2o secreted by s. sclerotiorum was only partially responsible for the observed decreases in photosynthesis. treatment with 40 mm k2c2o decreased fv/fm by about 70% of the levels observed under 40 mm h2c2o which further demonstrates that c2o42− was the primary factor that impaired photosynthetic performance during s. sclerotiorum infection. k2c2o treatment did not further decrease photosynthetic performance when d protein synthesis was fully inhibited, indicating that c2o42− inhibited psii by repressing d protein synthesis. it was observed that k2c2o treatment inhibited the rate of rubp regeneration and carboxylation efficiency. in the presence of a carbon assimilation inhibitor, k2c2o treatment did not further decrease photosynthetic performance, which infers that c2o42− inhibited psii activity partly by repressing the carbon assimilation. in addition, it was showed that c2o42− treatment inhibited the psii activity but not the psi activity.  this study demonstrated that the damage to the photosynthetic apparatus induced by s. sclerotiorum is not only caused by the acidity of h2c2o but also by c2o42− which plays a much more important role in damaging the photosynthetic apparatus. c2o42− inhibits psii activity, as well as the rate of rubp regeneration and carboxylation efficiency, leading to the over production of reactive oxygen species. by inhibiting the synthesis of d ros may further accelerate psii photoinhibition. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 