The implication of calcium as an early signal to abiotic and biotic stresses has been previously reported [12–16]. Here we have focused on the importance of calcium in relation to WIR in A. thaliana leaves. Wounded leaves produce a rapid and local burst of ROS and display a strong resistance when inoculated with B. cinerea. We have examined if changes in calcium levels are causally associated with ROS and WIR.
Our pharmacological approach using verapamil, EGTA or oxalate indicated a correlation between interference with calcium levels and loss of wound-induced ROS formation (Figure 1). These chemicals are known to act on calcium homeostasis and their application on leaves prior to wounding also inhibited WIR to B. cinerea (Figure 2). Overall, there was a correspondence between the concentrations at which these inhibitors interfered with ROS and those at which WIR was induced. Calcium is also required for basic resistance as non-wounded leaves treated with calcium inhibitors were also more susceptible to pathogen attack (Figure 2). The calcium channel blocker and chelating agents interfered with the early steps taking place after wounding and did not damage cells as indicated by the vital staining (Figure 1). Our results imply that calcium acts as an intermediary signal between the perception of wounding and WIR to B. cinerea. This conclusion supports previous observations on the importance of calcium in the process on induced resistance [13, 17].
But where is the effect of calcium exactly taking place? Wounding of leaves leads to an almost immediate crushing of cells with a loss of their integrity and compartmentalization. The calcium signalling for ROS formation and induction of WIR takes place in cells adjacent to the wound site . This would imply an almost instantaneous transmission of information from the crushed cells to their neighbours. Jasmonates are rapidly produced and translocated from cell to cell and across the plant . Although the experimental set-up used here is similar as in the report by Glauser et al. , jasmonates are unlikely to be involved, since mutants affected in jasmonate synthesis still express WIR ). Rapid signalling from the crushed cells to their neighbours might possibly be relayed by calcium itself: decompartmentalization after wounding leads to an instantaneous and local increase in calcium that will extend from the cytosol into the apoplasm. Verapamil or calcium chelators would act in preventing the calcium wave originating at the wounded sites to enter the intact neighbouring cells.
Is it possible to visualize changes in calcium levels in leaves after wounding? We have used plants expressing aequorin or Yellow Cameleon 3.6 proteins. Both types of reporter lines indicate changes in cytosolic calcium exclusively. Using both reporters it was possible to obtain reproducible kinetics of cytosolic calcium oscillations after wounding. A sharp peak occurred in the first seconds after wounding. This peak was transient and lasted only a few seconds. The second peak appeared around 30 seconds after wounding and it lasted ca 30 seconds longer than the first one (Figure 4). This calcium signature is difficult to interpret since it represents a view of a piece of tissue rather than single isolated cells. In fact, inhibitors or calcium chelators differently affected leaf tissues. Treatment with 100mM EGTA blocked both calcium spikes caused by wounding in the interveinal tissue, but it prevented exclusively the second peak in the veins (Figure 4). These results are in line with the observations using aequorin-expressing plants, where only the second peak is prevented by treatments with inhibitors or chelators of calcium (Figure 3). Aequorin-expressing plants are a much less precise method for our purposes: to obtain a detectable signal an entire leaf wounded on a wide surface should be analyzed and both interveinal tissue and veins are indiscriminately included in the quantification. The luminescence values read during this experiment are thus a sum of the single kinetics derived by the individual tissues. More importantly, aequorin is known to respond non-linearly in certain conditions and if the concentration of calcium is not homogeneous in the sampled population of cells, the overall aequorin light emission is dominated by the most responding sub-population . For those reasons the kinetics of calcium obtained by the Cameleon-expressing plants are more reliable and delimited to specific regions of interest giving a more precise idea of calcium dynamics after wounding. Both peaks are associated with the ROS formation and induction of WIR and their sensitivity to calcium inhibitors that act outside the cell would imply that calcium transits through the apoplasm.
Do the same group of cells that exhibit changes in calcium levels also produce a burst of ROS? FRET experiments combined with the introduction of the ROS marker DCF-DA showed changes in calcium levels in cells surrounding the wounded area accompanied by change in green fluorescence indicating a burst of ROS (Figure 5B). After the wound stimulus, the fluorescence caused by increased calcium levels was followed within minutes with the fluorescence reflecting increases in ROS, indication a) a colocalization of both processes and b) calcium changes precede ROS accumulation. These results obtained with cells embedded in the leaf are in agreement with many observations on cultured cells where calcium peaks preceded ROS . Our results on calcium changes preceding the formation of ROS at the same site are thus in agreement with our hypothesis. It is also interesting to note, that besides transient calcium and ROS production, the oxidative status of the cells surrounding the wounding site changes, most likely caused by the burst in ROS. These conditions could cause oxidation of various molecules, for example reactive cysteines present in the cell, hence potentially triggering other downstream responses . It was previously reported that a basal level of glutathione is required for WIR to B. cinerea. After a wounding event glutathione does not further accumulate in a Col-0 wild type, but wounding leads to the priming of the expression of a glutathione-S-transferase (GST1) gene. These results suggest that glutathione functions in detoxification during WIR rather than building up a reduction potential that might interfere with a ROS-driven cellular oxidation. However, given the cytotoxicity of a strong ROS burst, it cannot be excluded that glutathione might be still quench molecules arising form the oxidative burst. The role of ROS in WIR is not clear at the moment. Reports indicate a role of ROS in signalling , while others propose a model by which ROS play a role in cell wall reinforcing . We have already demonstrated that ROS are essential in WIR against B. cinerea.
What sensory proteins are possible targets for calcium? Calcium is a second messenger known to activate four different categories of calcium sensor proteins: calmodulins (CaMs), CaM-like proteins (CMLs), calcium-dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs) [23, 24]. Among the 34 CDPKs of the Arabidopsis genome , CDPK6 was recently reported to be involved in elicitor regulated ROS and innate immunity in protoplasts of A. thaliana. In potato CPK6, the ortholog of A. thaliana CPK5, can directly phosphorylate the membrane-bound NADPH oxidoreductase RBOH-D to stimulate its activity for ROS production in response to pathogens . However, our previous study has already excluded an involvement of RBOH-D in WIR . Here, we have tested cpk5, cpk6 and cpk11, single, double and triple mutants , but WIR was still active and the mutants were perfectly able to produce ROS after wounding (data not shown). Further work is now needed to identify components involved in sensing calcium.