3.2. Original OJIP Kinetics Normalized Relative to F0 (F0.02ms)In order to make it easier to analyze and more clearly represent the possible changes caused by the agents added to the control (in the absence of other additives); in comparison with the control, normalization is carried out to the initial level of fluorescence F0, as a rule, by the value of F20µs or F50µs measured at 20 µs or 50 µs, respectively [28], but sometimes by the F0 value measured at time t = 0 [45,46]. In recent years, normalization to F0.05ms has been favored, although normalization to F0.02ms is acceptable and still quite common [47,48]. In addition, it is shown that the possible errors in the calculation of the parameters of the JIP test in the case when F50µs is used as F0 is higher than for F20µs and Ft→0 [45].The original OJIP-kinetics normalized relative to F0 are presented as Ft − F0 versus time in Figure 3 (where F0 is the fluorescence at time 0.02 ms; Ft is the fluorescence at time t). The analysis of the presented kinetics shows the following main properties of the obtained kinetics and their changes caused by the studied agents and their combinations.Kinetics measured in the absence of additions (control) are completely identical to those recorded on PSII-containing membranes [42,43,44,49]. There is no peak I in the kinetic (plateau J–I), the main feature characterizing the kinetics of fast chlorophyll fluorescence induction measured on PSII-containing membranes [42,43,44,49] and therefore the kinetics will be designated below as OJP kinetics [42]. The absence of peak I (plateau J–I) in the OJP kinetics of PSII-containing membranes has been substantiated previously [42]. In the presence of both studied concentrations (3.6 μM and 14.5 μM) of [CuL2]Br2, a significant simultaneous almost synchronous decrease in the chlorophyll fluorescence intensity (F) is observed along the entire length of the OJP kinetics. The decrease also includes the FJ level (2–3 ms), and it is in greater extent in the presence of 14.5 μM [CuL2]Br2. The chlorophyll fluorescence decrease is especially significant at the FM level—in the presence of 3.6 μM and 14.5 μM [CuL2]Br2 by 22% and 45%, respectively, kinetics 2 and 3, Table 1 compared with the control (kinetic 1). The FM decrease is especially pronounced at 14.5 μM [CuL2]Br2 (kinetic 3). Let us designate these decreases in F (including FJ and FM) as described above as the “effect of [CuL2]Br2”.

Thus, these experimental data suggest that out of the total number of PSII-containing membranes, 22% and 45%, PSII-containing membranes (respectively, in the presence of 3.6 μM and 14.5 μM [CuL2]Br2) are no longer capable of photochemical reduction of the corresponding components of the acceptor side of PSII. This effect is a consequence of a certain suppressive effect of [CuL2]Br2 on the components providing either charge separation or the source of electrons from the components of the donor side of PSII, and onward can be excluded from further consideration because they no longer produce JIP kinetics due to the action of [CuL2]Br2. Therefore, the remainder of the total number of PSII-containing membranes that retained photochemical activity in the presence of 3.6 μM and 14.5 μM [CuL2]Br2, respectively, should be considered, namely 78% and 55%. And using these data, it will be possible to find out by what mechanisms [CuL2]Br2 disrupts the functioning of PSII and in what sequence these mechanisms function.

In addition, FM is reduced in the presence of 4 μM DCMU and especially in the presence of its combinations with both concentrations of [CuL2]Br2 (Figure 3, Table 1). Moreover, in the case of a combination of 14.5 μM [CuL2]Br2 + 4 μM DCMU, an almost synchronous decrease in the chlorophyll fluorescence intensity (F) occurs along the entire length of the OJP kinetics, which are similar to described above.In the presence of DCMU (without [CuL2]Br2), changes in the OJP kinetics characteristic of DCMU are observed (the so-called “DCMU effect”)—namely, an increase in the FJ peak to the so-called FM peak (kinetic 4), the intensity of which is less than the FM peak of control. The effects of DCMU have been repeatedly shown and explained previously by other authors [41,42,43,44,50]. In the presence of DCMU, all the amount of QA present in the sample is restored, which is expressed in an increase of the J peak to the highest possible level. At the same time, there is a decrease in the FM value to a value that is 62% from the control FM. This decrease is due to the quenching of F by oxidized PQ-9 molecules [41,42,43,44,50]. A further decrease in the FM intensity by above reason seems unlikely, since in a preliminary experiment, we showed that 4 μM DCMU inhibited the oxidation of all reduced QA molecules at the concentration of PSII-containing membranes we used.

Of particular interest and significance are the changes in OJP kinetics that occur in the presence of simultaneously both DCMU and [CuL2]Br2 (kinetics 5 and 6). Both kinetics are similar to the kinetics recorded in the presence of only DCMU (“DCMU effect” (kinetic 4)), but at the same time, the intensity of chlorophyll fluorescence decreases even more significantly over the entire OJP kinetics (“[CuL2]Br2) effect”). This decrease is especially evident in the case of 14.5 μM [CuL2]Br2)+ 4 μM DCMU (kinetic 6). The intensity of F at the FM level decreases in the case of these combinations of inhibitors (3.6 μM [CuL2]Br2) + 4 μM DCMU) and (14.5 μM [CuL2]Br2) + 4 μM DCMU), by 50% and 66%, respectively, relative to the control FM.

In this case, in the presence of both combinations of DCMU with 3.6 μM and 14.5 μM [CuL2]Br2 (similar to situation without DCMU described above), there is for further research only part from the total number of PSII-containing membranes that retained photochemical activity, namely 50% and 34% in this case relative to FM in the presence of 4 μM DCMU alone. In such case, in the presence of both combinations of DCMU with 3.6 μM and or 14.5 μM [CuL2]Br2, the remaining parts of the total number of PSII-membranes that retained photochemical activity, namely 50% and 34%, should be further considered.

Thus, DCMU induces a “DCMU effect” regardless of the presence of [CuL2]Br2. At the same time, [CuL2]Br2 effectively suppresses the FM value both in the absence and in the presence of DCMU.

In the presence of DCMU, it is important to correctly estimate how much [CuL2]Br2 reduces the FM value. Since a further decrease due to quenching of F by oxidized PQ-9 molecules remaining in PSII-membrane is unlikely, since oxidation of all available QA molecules is blocked by DCMU, then the observed decrease caused by both concentrations of [CuL2]Br2 in the presence of DCMU is based on another reason, and the percentage of decrease in FM in this case should be calculated by taking as 100% the value of FM measured in the presence of 4 μM DCMU. In this case, a further FM reduction due to quenching of F by oxidized PQ-9 molecules remaining in PSII-containing membranes is unlikely, since oxidation of all available QA molecules is blocked by DCMU. Consequently, the observed FM reduction caused by both concentrations of [CuL2]Br2 in the presence of DCMU is based on another reason. The percentage of decreased FM reduction in this case should be calculated by taking as 100% the value of FM measured in the presence of 4 μM DCMU. These calculated data are shown in Table 1 in parentheses and highlighted by asterisks. Comparing these data, we can see the following: in the absence of DCMU, both concentrations of [CuL2]Br2 suppress the FM value by 22% and 45%, respectively, and in the presence of DCMU, by 19% and 44%, respectively. These values are fairly well comparable.

The revealed coincidence of the values of FM decrease by [CuL2]Br2 in the presence of DCMU and without DCMU suggests that in both cases [CuL2]Br2 inhibits the activity of PSII-containing membranes by the same mechanism.

3.3. OJIP Kinetics Normalized Relative to F20µs and FMMany stresses, including high or low temperature stress; high light intensities; UV-B; inhibitors of PSII photochemical activity, etc., affect the photoinduced redox state of QA, and this is reflected in the form of changes in the intensity of the FJ peak of OJIP kinetics and/or time to J-peak [40,41,51,52,53].In Figure 3, it is not easy to understand how the intensity F changes at the level of peak J for almost every kinetic compared to the control, with the exception of kinetics 4 (4 μM DCMU) and 5 (3.6 μM [CuL2]Br2 + 4 μM DCMU) in which an increase in FJ intensity is clearly shown. Normalization of the original OJP kinetics simultaneously relative to the value of F0 and the value of FM makes it possible to reveal in more detail possible changes, including intermediate peaks, in the case of PSII-containing membranes—peak J. It was of interest to clarify more clearly how [CuL2]Br2 affects the properties of the J peak in the absence and the presence of DCMU.Figure 4 shows the original OJP kinetics normalized relative to F0.02ms and to FM. After such normalization, it became obvious that, in addition to the simultaneous decrease in the chlorophyll fluorescence intensity over the entire OJP kinetics (slightly at the FJ level (2–3 ms) and especially pronounced at the FM level), which was clearly pronounced after normalization original OJP kinetics relative only to F0.02ms, now there are significant changes in OJP kinetics compared with the control in the presence of both concentrations of [CuL2]Br2, as well as their combinations with DCMU, which in this case became especially pronounced in the region of peak J (Figure 4).From the data presented in Figure 4, it is evident that: (1) without DCMU in the presence of 3.6 μM [CuL2]Br2 (kinetic 2), the intensity of the J peak increases compared to the control (kinetic 1), but at a higher concentration of [CuL2]Br2 (14.5 μM) (kinetic 3), this effect, which is expressed in an increase in the J peak, already becomes significantly less; (2) in the case of a combination of 3.6 μM [CuL2]Br2 and 4 μM DCMU (kinetic 5), the J peak becomes a little bit higher compared to 4 μM DCMU (kinetic 4), however, at a higher concentration of [CuL2]Br2 (14.5 μM) in this combination inhibitors (kinetic 6), a significant decrease in the J peak is already observed.

Thus, in both above cases (namely in the absence and in the presence of DCMU), the differently directed effect on the F intensity of the J peak of these two concentrations of [CuL2]Br2 (3.6 μM and 14.5 μM) is clearly visible. It should emphasize that in the presence of DCMU the difference in the above effects between these concentrations is much greater. Despite the fact that after this normalization it is possible to identify additional changes in the OJP kinetics, nevertheless, in this case, these changes are not yet clearly expressed, and it is not possible to quantify the degree of these changes.

3.4. Comparison [CuL2]Br2 and DCMU EffectsPeak I is known to be absent in PSII-containing membranes [34,42,43,44,49]. The IP phase is directly related to PSI activity, while JI phase parallels the reduction of PQ pool [27,28].Since there is no I peak in PSII-containing membranes, in order to more conveniently analyze and visualize possible changes at the level of the J peak, which are induced by the studied inhibitory agents and their combinations, we first double normalized the original kinetics relative to both the F0 level (F0.02ms) and to the level of finding the peak I (30ms), i.e., to the level F30ms, according to the formula:

V0I = (Ft − F0)/(FI − F0),

in our case

V0I = (Ft − F0.02ms)/(F30ms − F0.02ms)

The resulting kinetics V0I = (Ft − F0.02ms)/(F30ms − F0.02ms) are shown in Figure 5A. Next, we subtracted the kinetic obtained in the absence of any additions (control) from the kinetics obtained in the presence of inhibitory agents, for each of the studied inhibitory agents and their combinations. The obtained difference kinetics W0I = V0I experiment − V0I control are shown in Figure 5B.It is known that DCMU blocks electron transfer from the reduced primary PSII electron acceptor, plastoquinone QA, into the membrane pool of plastoquinones (PQ-9), competing with the PSII secondary electron acceptor, plastoquinone QB for the binding site on the so-called QB herbicide-binding site of the D1 protein. Therefore, in the presence of DCMU, the so-called “diuron effect” is observed, which is expressed on the original OJIP kinetics as a significant increase in fluorescence intensity at a level of 2–3 ms (peak J) compared to the control [40,41,51,52,53]. This can be especially clearly seen in the difference OJP-kinetics obtained by subtracting from OJP-kinetics measured in the presence of DCMU, the kinetics obtained in the absence of any additions (control) [52,53].

Preliminarily, for the conditions of our measurements (the concentration of PSII-containing membranes, expressed as the concentration of chlorophyll contained in them, is 4 μg mL−1), we found that the concentration of DCMU used by us (4 μM) causes practically maximal “diuron effect” on the chlorophyll fluorescence of PSII.

In addition, the use of higher concentrations of DCMU may be accompanied by the effects of DCMU on other sites of the PSII electron transport chain, as described earlier [54,55,56,57]. In order to quantify the “diuron effect” of other studied inhibitory agents or their combined use with DCMU, we evaluated the FJ values for the other difference OJP-kinetics (W0I = V0I exp − V0I control) presented in Figure 5B in % from that with DCMU. The magnitude of the “diuron effect” FJ measured in the presence of 4 μM DCMU, which indicates amount of reduced QA (QA−), we took as 100%. And the effects of other supplements were evaluated in % relative to this effect of DCMU. The data obtained are presented in Table 2. 3.4.1. Effects of [CuL2]Br2 in the Absence of DCMUFrom the data presented in Table 2, it can be seen that in the absence of DCMU, low concentrations (3.6 μM) of [CuL2]Br2 cause a “diuron effect” of approximately 38% of that caused by DCMU. With an increase in the [CuL2]Br2 concentration to 14.5 µM, the “diuron effect” increases and is already 71% of the “diuron effect” caused by DCMU. 3.4.2. Effects of [CuL2]Br2 in the Presence of DCMU

A completely different effect of the [CuL2]Br2 complex on the photochemical activity of PSII is observed when [CuL2]Br2 complex is added in the presence of DCMU. In this case, both concentrations (3.6 μM and 14.5 μM) of [CuL2]Br2 significantly reduced the “diuron effect” of DCMU from 100%, respectively, to 59% and to about 3%.

Thus, from the data presented in Table 2, it is obvious that in the absence of DCMU, the amount of reduced QA increases with increasing concentration of the [CuL2]Br2 complex. However, in the presence of DCMU, on the contrary, the amount of reduced QA decreases significantly with an increase in the concentration of the [CuL2]Br2.

We evaluated the potency of these effects of [CuL2]Br2, namely (1) the effect of increasing the amount of reduced QA in the absence of DCMU and (2) the effect of decreasing the amount of reduced QA in the presence of DCMU on the concentration of the [CuL2]Br2 complex from the slope of the corresponding fitted curves. It turned out that the second mechanism of action of the [CuL2]Br2 complex, which manifests itself in a decrease in the amount of reduced QA in the presence of DCMU, is about two times more effective than the first one, the accumulation of the amount of reduced QA in the absence of DCMU.

Based on the comparison of the positions of the J peaks on the time scale, it can be roughly assumed that in the presence of 4 μM DCMU, the time to reach the maximum value of the fluorescence intensity of the J peak (FJ) on the difference kinetics WOI = VOI exp − VOI control (Figure 5B), which characterizes the rate of QA reduction with increasing concentration of the [CuL2]Br2 complex, also increases—as can be seen when comparing the difference kinetics for (3.6 μM [CuL2]Br2 + 4 μM DCMU) and (14.5 μM [CuL2]Br2 + 4 μM DCMU). Moreover, this property of the [CuL2]Br2 complex to slow down the rate of photoinduced QA reduction even increases with an increase in its concentration with DCMU. This is evident when comparing the different kinetics of (3.6 µM [CuL2]Br2) and (3.6 µM [CuL2]Br2 + 4 µM DCMU), as well as (14.5 µM [CuL2]Br2) and (14.5 µM [CuL2]Br2 + 4 µM DCMU).

However, there is a more reliable way to quantify the rate of photoinduced QA reduction.

3.5. Estimation of the Rate of Photoinduced Reduction of QAGraphical or computational determination of the initial slope (M0) of the JIP kinetics makes it possible to estimate the rate of photoinduced reduction of QA and its changes as a result of various influences [53,58,59,60,61,62]. We have used both of these approaches.The graphical data presented in Figure 6 allow you to see much more clearly what changes are induced by the studied agents in PSII photochemical reactions; the graphical approach is also used by other researchers [53,58,60,62]. In addition, we calculated the values of M0 using the corresponding formula M0 = 4 (F300 − F0)/(FM − F0). The results of the calculations are presented in the Table 3.It should be noted that the values (M0) determined on the basis of the data presented in Figure 6 almost coincide with those obtained as a result of calculations. However, since the “first” ones were determined as a result of approximating real values, the data obtained in the calculations should be considered more accurate.From the data presented in the form of kinetics in Figure 6 and the corresponding values of M0 in the Table 3, it follows that in all variants in the presence of the studied agents (both concentrations of [CuL2]Br2 without DCMU, 4 μM DCMU, both combinations of [CuL2]Br2 with DCMU), the rate of accumulation of reduced QA (QA−), compared to the control is above (Figure 6 and Table 3).

If we evaluate the rate of accumulation of reduced QA (QA−), compared with DCMU, then in the absence of DCMU, both concentrations of [CuL2]Br2 increase the rate of accumulation of reduced QA (QA−), compared with the control, as well as in the presence of DCMU alone, however, with a significantly lower efficiency compared to DCMU (46% and 32% of that of DCMU) (kinetics 4 and 5). Interestingly, in the presence of a lower concentration of [CuL2]Br2 (3.6 µM), this effect is greater (46%) compared with a higher concentration (14.5 µM) of this agent (32%), i.e., without DCMU, the ability to cause an increase in the rate of QA reduction decreases with increasing concentration of [CuL2]Br2.

In the presence of DCMU, [CuL2]Br2 also reduces the rate of accumulation of reduced QA (QA−), respectively, to 69% and 56%, relative to DCMU (Table 3 of kinetics 2 and 3), and this effect of [CuL2]Br2 also increases with increasing concentration [CuL2]Br2.We evaluated the effectiveness of the impact of [CuL2]Br2 on the rate of QA reduction in the absence and presence of DCMU by the slope of the approximated lines plotted using the corresponding experimental data from Table 3. It turned out that both in the absence of DCMU and with DCMU, the rate of QA reduction with increasing concentration of [CuL2]Br2 goes down. However, in the presence of DCMU, the rate of QA reduction with increasing concentration of [CuL2]Br2 decreases approximately three times faster than in the absence of DCMU.