Biochemical analysis performed on chloroplasts isolated from studied plants seedlings allowed detecting the differences in ability of these organelles to protect plants against oxidative stress resulted from ZEN accumulation, depending on stress tolerance of wheat varieties and the way of application of this mycotoxin. Higher amounts of superoxide radicals observed in chloroplasts of the sensitive variety versus tolerant might indicate less efficient action of antioxidant system in this variety. It was interesting, that this phenomena was observed in samples regardless of the method of application and subsequent transport of ZEN to chloroplasts. Better potential antioxidant properties of chloroplasts of the tolerant cultivar compared to sensitive one were confirmed by higher values (under control conditions) of all tested indicators (DPPH, AA, Fe(III)/Fe(II)).
DPPH appears in the presence of hydrogen donating antioxidants in the non-radical form . The decrease of scavenging activity upon ZEN treatment could result from involvement of antioxidants in removal of ROS generated under stress. The greater decrease associated with long-term ZEN-stress (application to grains) could be caused by more effective synthesis of antioxidants than after short-term interaction of ZEN (foliar treatment), similarly as it was observed in microscopic visualization of superoxides.
Ascorbic acid (AA) takes part as a factor in redox reactions in which one reactive substance is reduced at the expense of the oxidation of another . Also the values of “reducing power” expressed as the ability of the reduction Fe(III) ions to Fe(II) indicated that redox reactions in chloroplasts under ZEN application played an important role in defense processes. A higher content of Fe(III) ions in control samples of tolerant plants was connected with the lower level of free electrons that might reduce these ions to Fe(II) and pointed to more effective action of the antioxidative system of this variety. The decrease of Fe(III) amounts, caused by ZEN application (greater in case of foliar treatment than when grains were ZEN infected) indicated the increase of free electrons amounts, generated in stress conditions. The decrease of reducing power was bigger in samples originating from sensitive wheat genotypes, confirming that antioxidant system was less efficient in this genotype.
The studies of EPR signals, despite the fact that the measurements were not performed directly on chloroplasts, allowed characterization of paramagnetic metal ions whose main locations and functions were associated with these organelles. Moreover, changes in redox status of metal ions upon ZEN treatment could be monitored. The analysis of parameters of EPR signals appearing in spectra of studied plant material combined with literature data allowed attribution of observed signals to particular paramagnetic species. The signal of six hyperfine lines overlapping a broad one was ascribed to Mn species. Well resolved hyperfine structure observed at room temperature originated from freely rotating aqua - complex of Mn(II) and was often found in EPR spectra of various plants [6, 10], while the broad line was ascribed to dipole - dipole interacting Mn(II) ions situated mainly in protein matrix . Signals observed in g range between 2.2–2.5 were attributed to inorganic antiferromagnetically coupled paramagnetic Fe(III) ions forming Fe-O-Fe clusters, ferric oxides, oxyhydroxides and/or phosphates which were accumulated in the “iron-core” of ferritin [18, 19], whereas broad signals at g = 2.4 and 2.6, appearing at 77 K, were ascribed to Fe(III) ions bonded to protein matrix in the ferritin protein shell, containing ferric and ferrous ions [20, 21]. The small line with g = 4.26, observed in all spectra recorded at 77 K, was attributed to non-hem high spin Fe(III) with rhombic symetry .
Calculation of intensity of particular signals, recorded at room temperature, indicated that at control conditions, signal of Mn(II) ions had a significant contribution to the spectrum, especially for Parabola, in which its intensity was about 4 times bigger than that of Fe(III), while for Raweta only about 2.5 times (Fig. 5A’, A”). Upon ZEN treatment the observed increase of integral spectrum intensity of Parabola leaves (Fig. 5A), measured at room temperature, was caused mainly by changes of manganese signal, whereas for Raweta genotype it resulted from the growth of Fe(III) signals at g in the range 2.2–2.5 (Fig. 3B, 5A”). The stronger increase of signal intensity of manganese aqua complexes for Parabola than for Raweta, treated with ZEN independently of application way (Fig. 5A’, A”), could suggest better accumulation of water in tissues of tolerant genotype. The binding of water molecules in plant tissues may be considered as one of factors in the cell stress protection ensuring the appropriate quaternary structure of proteins participating in photosynthesis processes. In sensitive genotype growing intensity of Fe(III) signal could result from oxidation of Fe(II) species by ROS, generated during ZEN action (Fig. 3B, 5A’, A”). At applied EPR measurement conditions Fe(II) ions are invisible in spectra. The strong line at g = 2.06 and four lines around g = 2.4, situated in the place of Fe(III) signals, observed in the spectrum of leaves of Raweta directly treated with ZEN, were ascribed to Cu(II) ions in square planar complexes of proteins  (Fig. 3C). Hence, it could be suggested that Cu(I) species, silent in EPR, were probably present in untreated plant material and underwent oxidation to Cu(II) upon ROS, similarly as it was observed for Fe species.
In the spectra of control plants measured at 77 K, contrary to room temperature, the signals of Fe(III) ions with g equal to 2.4 and 2.6 were dominant (Fig. 5B”) and two times higher in spectra of Parabola genotype. Upon ZEN treatment the intensity of these signals decreased, whereas that of Mn(II) signals increased for both varieties. The changes of Fe(III) signals were more visible for Parabola when grains were ZEN-infected, whereas for Raweta these changes were smaller. In the latter, Fe(III) signal (g = 2.4) in the spectrum of ZEN treated leaves overlapped Cu(II) signal (Fig. 3F, 5B”), hence the intensity of the line was a sum of those originating from Fe and Cu species.
Decrease upon ZEN of amount of Fe(III) species in plant tissues (Fig. 5B’, B”) resulted probably from the degradation of Fe(III) - protein complexes and was observed for all plants treated with ZEN, regardless of the way of its introduction (directly or indirectly) confirming that ZEN exerted negative influence on plants. The amount of Fe(III) ions present in plants subjected to ZEN treatment was the result of two processes: degradation of ferric protein complexes (this process was visible in the spectra recorded at 77 K) and oxidation of ferrous ions probably by ROS generated upon ZEN stress (observed in the spectra measured at 293 K). The observed phenomenon indicated that the way of ZEN treatment influenced the mechanism of damage of plant structures.
The changes of signals originating from radical species can provide further data on the influence of ZEN on wheat plants. Both overlapping signals, A and B, giving line R recorded at room temperature in spectra of control samples, were characteristic for carbon centered radicals located in carbohydrate molecules [5, 21]. The changes in intensities of these signals upon ZEN treatment were connected with modification of the signal R by the appearance of additional lines situated symetrically around line R (Fig. 4B, C), which were more intensive in spectra of leaves directly treated with ZEN. The lower value of g factor of signal C could indicate that when ZEN interacted directly with leaves, radicals were created at carbohydrate molecules with lower molecular weight, whereas the species created in plants grown from infected grains, giving signals with higher g factor, were situated at carbohydrates with higher molecular weight, for example in starch.
Intensity of signal R measured at room temperature was slightly lower for Raweta control plants than Parabola ones. ZEN treatment of grains decreased it, whereas application to leaves increased its value, more visibly in Raweta (Fig. 6A). The creation of stable carbohydrate radicals occurred as a result of transfer of free electrons from ROS, formed upon ZEN action, to carbohydrate molecules. Such stabilization of electrons, more effective in Raweta, was one of the defense mechanisms of plant against stress, as it was postulated by our earlier hypothesis, assuming that short direct stress led to the increase of the amount of stable carbohydrate radicals mainly in sensitive genotypes, whereas in tolerant ones other mechanisms were activated .
A new signal R’, recorded at 77 K (Fig. 4D-F), observed in many plant systems was ascribed to the stable tyrosyl radical , acting as an electron transfer in biochemical redox processes . Its amount increased strongly in leaves being in the direct contact with ZEN, simultaneously with increasing g1 parameter to 2.0091, suggesting that upon ZEN treatment the disturbance of tyrosyl radical geometry occurred. It was probably caused by removal of the hydrogen atom from the hydroxyl group of the tyrosine molecule [19, 22] by reactive oxygen species formed upon ZEN stress. It pointed to destruction of organic matrices, as it was indicated by disorder in biochemical surroundings of metal species, more noticeable in sensitive genotype. The lack of significant alterations in tyrosyl radical concentration in plants grown from grains treated with ZEN was in line with observed small changes in amounts of carbohydrate radicals and could indicate that if more time elapsed from the moment of contact with ZEN, the redox equilibrium in plant tissues was re-established, whereas after direct stress processes of radical transformation continually occurred.