3.1 Fumonisin B1 induces rapid cell death in Arabidopsis cell cultures
Previously, we have shown that Arabidopsis cell culture is a good model for studying natural senescence and induced programmed cell death (PCD), namely by high concentration of cytokinins (Carimi et al., 2004; Carimi et al., 2005) and heavy metals (De Michele et al., 2009). Under normal subculturing conditions, cells experience an exponential growth phase for the first 10 days, followed by a stationary lag phase and an eventual decline due to starvation (Carimi et al., 2005). In order to study the effect of FB1 in Arabidopsis cell cultures, we treated cells at the beginning of their linear growth phase, when they are at their best healthy conditions.
Arabidopsis cells suspension cultures were treated with two different FB1 concentrations, 1 and 5 µM. Mock-treated control cells maintained a linear growth pattern as assessed by fresh weight measurements, doubling between one and three days from treatment (Fig. 1 a). Cells treated with FB1 showed a marked reduction in growth, which was more severe in the 5 µM treatment. Four days after treatment, cells with 5 µM FB1 weighted less than half of controls. To determine whether the impairment in growth was an induction of lag phase or rather depended on increased mortality, we quantified dead cells. Whereas control cells showed a physiological 10% rate of dead cells along all the experiments, as expected from their growth curve (Carimi et al., 2005), cells treated with FB1 dramatically increased their mortality (Fig. 1 b). Cell death increased as early as 1 day after treatment with 5 µM FB1, and eventually reached 45%.
In plants, cell death may be characterized by a wide range of features, from necrosis to full PCD. A typical marker of PCD, especially the “slow” events such as natural and induced senescence, is the gradual condensation of DNA within the nuclei, often appearing with a sickle shape, as opposed to the relaxed and round aspect of healthy nuclei. The following event during PCD is the cleavage of DNA in the inter-histonic spaces, leading to a ladder band pattern after electrophoresis (Carimi et al., 2005; De Michele et al., 2009). Conversely, necrosis or “fast” PCD events such as the HR often present a chaotic degradation of the DNA molecules, resulting in a smear after electrophoresis. In order to determine whether FB1-induced cell death showed typical PCD hallmarks, we checked DNA integrity by looking at its fragmentation pattern and nuclear condensation. When run in a gel, DNA from control cells was intact, as indicated by the high molecular weight band (Fig 1 c). Conversely, treatment with 5 µM FB1 resulted in an eventual DNA degradation in a smear, in agreement with the rapid and potent toxic effect observed in cell death measurements. As a further test for characterizing FB1-induced cell death, we analyzed the expression of SAG12, a well-known specific marker for senescence, induced during both natural and induced senescence in Arabidopsis cell cultures (Carimi et al., 2005; De Michele et al., 2009). FB1-treated cells, as well as healthy control cells, never showed SAG12 induction (Fig. 1 d), suggesting that the cell death event did not resemble an accelerated senescence, thus differing from other PCD inducers such as BAP and cadmium (Carimi et al., 2005; De Michele et al., 2009). On the other hand, several nuclei of FB1-treated cells showed sickled condensed nuclei when looked at the microscope, as opposed to control cells (Fig. 1 e). Nuclear condensation is present in rapid PCD processes such as the HR triggered by pathogens. Since FB1 is a mycotoxin produced by a plant pathogen, it is likely that the cell death caused by FB1 treatment resembles a HR-like PCD event. In agreement with our observation, Asai and colleagues already had observed typical PCD markers such as positive TUNEL nuclei in Arabidopsis protoplasts treated with FB1 (Asai et al., 2000).
3.2 Fumonisin B1 induces an oxidative and nitrosative burst
It is well known that in plants the HR response caused by an incompatible pathogen interaction is characterized by an early oxidative and nitrosative burst (Romero-puertas et al., 2004). In particular, hydrogen peroxide (H2O2) and nitric oxide (NO) are two players identified first in HR. Yet, the chemistry and the crosstalk among the different members of reactive oxygen and nitrogen species (ROS and RNS) is complex, and may differ greatly depending on the concentration, timing and localization of each molecule. ROS comprise the above mentioned H2O2 but also the superoxide anion (O2-), hydroxyl radicals (.OH) and singlet oxygen (1O2), produced during electron transport chains in chloroplasts and mitochondria, or by oxidases and peroxidases in peroxisomes and in the apoplast. RNS, besides the well-known NO, include the peroxynitrite anion (ONOO-), which forms by reaction of NO with O2-.
To add on the complexity of the crosstalk among these players, it is known that NO and H2O2 can interact to promote the formation of .OH and 1O2, but NO can also scavenge H2O2, thus protecting plant cells from damage. In order to assess whether FB1 treatment, by mimicking an HR response, caused and oxidative and/or nitrosative burst, we measured ROS and RNS production along time. Since ROS and RNS can act as signaling molecules, as well as late downstream cell death effectors, we extended our analysis as early as 1 h after treatment, focusing with 5 µM FB1 concentration, which gave the strongest response in the cell physiology assays. As a generic measure of oxidative stress, the levels of the fluorescence dye H2DCF-DA maintained at the same level of control cells for the first six hours. At 24 h, and even more at 48 h after treatment, cells experienced a high level of oxidative stress (Fig. 2a). Looking at the specific reactive species involved, we observed that the extracellular H2O2 release, as well as intracellular O2- levels, were late events, being significantly higher than control only after one day of treatment (Fig 2b, c). Conversely, NO and ONOO-/.OH increased as early as 24h after FB1 exposure (Fig 2d, e). Being ONOO- produced as result of reaction between NO and O2- , it comes with no surprise that its pattern followed those of the parent species. It is interesting to notice that an early NO production, preceding H2O2, was similarly observed in Arabidopsis cell cultures treated with the heavy metal cadmium, and it was causally linked to the onset of programmed cell death (De Michele et al 2009). It is tempting to speculate that the concomitant presence of NO, H2O2, and possibly other ROS and RNS species, is therefore a general feature of programmed cell death in plants.
In addition, intracellular H2O2 was evaluated. Its level was significantly higher than the control for both treatments during all the assay, with FB1 1µM having the utmost effect (data not shown).
To assess the effect of the oxidative and nitrosative damage, the level of lipid peroxidation was measured. A significant higher level of MDA content at 6 h of the treatment was observed indicating increased oxidative status of cell membrane. An unexpected significantly lower MDA level was found at 24 h and 72 h for FB1 5µM and at 72 h for FB1 1µM, as compared to control cells (Fig. 2f).
A possible explanation could be higher GSH level found in the FB1 treated-cells when compared with control (see Section 3.4); GSH can prevent damage to important cellular components as membranes caused by reactive oxygen species. It is able to reach directly, free radicals, peroxides, lipid peroxides, and heavy metals and is involved in pathogen resistance (Noctor and Foyer, 1998). Indeed, GSH differs from other metabolites that may play a similar role because of the presence of specific enzymes that link GSH with H2O2 metabolism, the stability of the corresponding oxidized form, and the ability to be recycled to reduced form through a powerful enzymatic system that depends on the electron transport molecule NAD(P)H (Foyer and Noctor, 2011).
3.3 Differential modulation of cell death responsive genes during FB1 exposure
To verify whether a defense response took place under FB1 treatment, the transcriptional changes of a set of genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, resistance and sugar transport were monitored at 24 and 48 h after exposure in Arabidopsis cells (Fig. 3-5). Considering the previously assessed cell growth pattern by measurement of fresh weight and mortality, as well as the pattern of ROS and RNS production, these two time-points were selected as the most relevant to decipher the early molecular changes produced by the mycotoxin. Moreover, we included the 1µM FB1 concentration in these analyses, to evaluate the differences between a strong and a weak dose of toxin. The relative expression profiles were calculated as fold change (FC) of FB1 treated over mock-treated cells.
Regarding the genes associated with the ageing processes and PCD control, all assayed genes were upregulated considering both FB1 concentrations and times of treatment (Fig 3). Exceptions were observed for the long chain bases 2b (LCB2b) gene at 24 h after 1 and 5 µM FB1 exposure (FC of -1.2 and -1.1, respectively; Fig 3 f). Drosophila DIAP1 like 1 (DAL1) showed the highest induction values at 24 h for both concentrations with expression levels of about 9 and 10 after 1 and 5 µM FB1 treatment, respectively (Fig 3 b). Similar transcriptional profiles were observed for the senescence-associated gene 21 (SAG21) that significantly peaked at the same conditions (FC of about 5), followed by a decline at the later time of treatment (Fig 3 a). An opposite trend was detected for the other genes that reached a more marked upregulation almost always at 48 h after 5 µM FB1 treatment. This was more accentuated for the genes DAL2, the inhibitor of apoptosis protein (IAP) and LCB2a (Fig 3 c, d and e).
SAG21 belongs to the late embryogenesis-associated (LEA) protein family and, despite being first identified as early senescence-associated gene (Hundertmark and Hincha, 2008), it is also induced by H2O2 and superoxide (O2•)-donors and pathogen infection (Mowla et al., 2006; Salleh et al., 2011), thus constituting a general PCD marker. Additionally, the implication of SAG21 in response to mycotoxin treatment in plant cells was reported in several works. Wang et al. (2012) described higher transcript levels for SAG21 along with additional senescence-activated genes, SAG13 and SAG18, and the senescence-related gene SAG2 8 h after ochratoxin A (OTA) treatment in Arabidopsis leaves. Similarly, FB1 exposure for a time course of 20 h stimulated SAG21 induction in Arabidopsis protoplasts (Asai et al., 2000), confirming the involvement of this gene relatively shortly during PCD. SAG21 induction contrasts with SAG12, which was not induced by FB1, nor in young control cells (Fig 1D). The SAG12 papain-like cysteine protease is, so far, the best known senescence marker, being strongly induced in senescent leaves of Brassica napus L. and A. thaliana, especially in plants cultivated under nitrogen limitation (Desclos et al., 2008; Poret et al., 2016). Moreover, elevated SAG12 protein levels were measured in senescing leaf tissues and fallen leaves (Desclos-Théveniau et al., 2015). Nevertheless, studies carried out on sag12 mutants did not reveal any differences in phenotypic traits and leaf senescence progression compared to wild type plants (Otegui et al., 2005. Additionally, the lack of SAG12 was not harmful to the formation of senescence-associated vacuoles and the ribulose-1,5-bisphosphate carboxylase/oxygenase degradation (Otegui et al., 2005). Overall, SAG12 is therefore a good marker of senescence, although it is functionally not necessary to its progression. Since SAG genes encode for a wide family of proteases showing a broad range of sequence diversity, intracellular localizations, and expression patterns, it could be supposed that other proteases, including SAG21, could counterbalance the impaired expression and activity of SAG12 during senescence and other PCD events, such as FB1-elicited toxicity.
DAL1 and DAL2, two RING finger proteins homologous to Drosophila DIAP1, are functional negative regulators of PCD in Arabidopsis. A previous study showed that dal1 and dal2 mutants significantly accumulated superoxide anions, determining PCD after the inoculation of Arabidopsis leaves with Pseudomonas syringae pv. tomato (Pst) (Basnayake et al., 2011). Furthermore, the expression of DAL1 and DAL2 genes was abundantly increased after Pst and 10 µM FB1 treatment in wild-type plants with the highest induction at 42 h (Basnayake et al., 2011). These results are in line with those obtained from this work, since we also observed induction after FB1 treatments, though the peak timing and expression change intensity varied between DAL1 and DAL2 (Fig. 3).
Besides the DAL ring finger proteins, a further ring finger protein, the Arabidopsis inhibitor of apoptosis IAP showed its implication in the protection against cell death preventing caspase activation. This was pointed out by Kim et al. (2011), which reported a strong anti-apoptotic activity in transgenic Arabidopsis plants overexpressing IAP when treated with FB1. Furthermore, the inhibition of DNA fragmentation and caspase activity as well as an attenuated cell death caused by the bacterial effector AvrRpt2 was observed in the same plants, confirming the role of IAP as negative regulator of PCD in Arabidopsis (Kim et al., 2011).
Sphingolipid long-chain bases (LCBs) represent crucial PCD mediators in plants. The relationship between FB1 and sphingolipid pathway was previously demonstrated using Arabidopsis deletion mutants (Shi et al., 2007; Saucedo-Garcia et al., 2011; Kimberlin et al., 2013; Shao et al, 2020). More in detail, the insertional mutant FB1-resistant 11 (Fbr11) characterized by a deletion in the gene encoding for a LCB1 subunit of serine palmitoyltransferase (SPT) displayed lower levels of LCBs, but improved tolerance to FB1 (Shi et al., 2007; Kimberlin et al., 2013). Similarly, lcb2a mutants were unable to rise an effective PCD after 10 µM FB1 exposure, highlighting that the gene LCB2a is essential for PCD elicitation (Saucedo-Garcia et al., 2011). Furthermore, the fbr41 mutants overexpressing the LCB2b gene exhibited less severe cell death phenotype when challenged with FB1 and Alternaria toxins (Shao et al, 2020). LCBs are also involved in the mitogen-activated protein kinase (MAPK) cascade. Saucedo-Garcia et al. (2011) demonstrated how MAPK6 was activated in response to FB1 and behaved as a transducer during the LCB-induced PCD. The enhanced transcript accumulation observed in this study for the genes LCB2a and b, and MAPK6, predominantly at the later time of incubation (48 h) and at higher concentration of FB1 (5 µM), confirm the contribution of sphingolipid pathway to the cytotoxicity of this mycotoxin in Arabidopsis cells too.
The expression profiles of the antioxidant genes ascorbate peroxidase (APX) and respiratory burst homologue C (AtrbohC), the aminocyclopropanecarboxylate (ACC) oxidase involved in the ethylene production, the phosphoglycerate kinase (PGK), the serine hydromethyltransferase 1 (SHM1) and the pheophytnase (CRN1), related to the photosynthetic and photorespiration processes, respectively, were also analyzed in this work (Fig 4). In general, these genes showed a higher transcript accumulation during the late treatment time, more enhanced at 5 µM concentration namely for the ACC oxidase, PGK and SHM1 (Fig 4 c-e). No significant variation was displayed by the APX and CRN1 genes for both treatment times and concentrations, except CRN1 at 48 h that resulted significantly more expressed under 5 µM FB1 exposure (Fig 4 f).
It is known that ethylene (ET) is involved in plant responses to FB1 and contributes to PCD and activation of defense mechanisms by a concentration and time-dependent manner (Zeng et al., 2020; Iqbal et al., 2021). Different phenotypes were observed in the Arabidopsis ethylene response 1-1 (etr1-1) mutants, probably due to the diverse light and growth conditions (Asai et al., 2000; Iqbal et al., 2021). Wu and co-workers (2015) by employing several ET mutants reported that sphingolipid synthesis was suppressed by ET signaling that acted as a negative regulator of FB1-challenged PCD. Moore et al. (1999) showed that 0.1 µM FB1 treatment of tomato leaflets determined an enhanced transcript accumulation of ACC synthase and ACC oxidase in the late times of exposure, in line with our findings. The increase in ACC oxidase transcript was supported by co-occurring ASC increases, the latter acting as a cofactor of the enzyme and therefore involved in the synthesis of the hormone ethylene (Smirnoff, 2018). However, further research regarding the analysis of additional genes will contribute to clarify the role of this hormone in the FB1-induced cell death. ET also affects PCD via ROS accumulation. In this regard, it was found that FB1 (10 µM) elicitation rapidly induced ROS production in Arabidopsis leaves already after 3 days (Xing et al., 2013). Interestingly, in a further study, albeit Arabidopsis leaves infiltrated with FB1 exhibited high ROS production within 24 h, the expression of three antioxidant genes catalase, APX and peroxidase was not affected. In contrast, the transcript levels of AtrbohD and F slightly accumulated at 48 h in the same conditions (Qin et al., 2017). Furthermore, Wang et al. (2012) described an increased upregulation of AtrbohC, the same gene analyzed in this study, AtrbohD and APX after OTA treatment of excised Arabidopsis leaves in the first 24 h. Additional experiments focusing on different Atrboh isoforms and more antioxidant enzyme-coding sequences will clarify our findings more accurately in light of these previous studies.
ROS generation is greatly influenced by chloroplast metabolism and active photosynthesis. Defense responses against mycotoxins are often light dependent and this was earlier reported for OTA, FB1, and deoxynivalenol (DON) (Wang et al., 2012; Xing et al, 2013; Ansari et al., 2014). Agreeing with our outcomes, the expression of CRN1 gene involved in the process of chlorophyll degradation was reported to be strongly induced under OTA stress (Wang et al., 2012). Conversely, SHM1 and PGK, essentials for the C2 cycle photorespiration and carbon dioxide fixation, respectively, were suppressed (Wang et al., 2012); while in this work, they were activated of about three times at 48 h after 5 µM FB1 treatment. Future investigations should examine more in depth the relationship between light regulated pathways and PCD in response to the mycotoxin FB1.
FB1 also determined the induction of four pathogenesis-related genes, PR1, PR2, PR5 and PR6 (Fig 5). Interestingly, the maximal transcript accumulation for all PR genes was measured after 48 h of treatment with FB1 at 1 µM (average FC of about 16), whereas a down-regulation was observed for both concentrations at the earlier time (Fig 5 a-d).
The elevated expression of PR genes upon FB1 exposure was previously described in several studies. Stone et al. (2000) reported that FB1 elicited PR1, PR2 and PR5 induction and this trend was directly proportional to the mycotoxin concentration (0.01-1 µM). Similarly, Arabidopsis leaves infiltrated with 10 µM FB1 exhibited an elevated expression for the same genes next to PR3 and the jasmonic acid-related PDF1.2 response gene (Zhang et al., 2015). The accumulation of PR1 and PR5 transcripts was also found in the same material by Qin et al. (2017), along with ROS and salicylic acid accumulation as well as lesion formation. These two genes were strongly induced in Arabidopsis leaves after OTA exposure too (Wang et al., 2012). The significant role of PR genes was described in other species besides Arabidopsis, as tomato plants and maize embryos. Accordingly, the overexpression of the gene P14a, a member of the PR1 family, prevented FB1-induced PCD in tomato roots (Lincoln et al., 2018). Furthermore, FB1 treatment positively modulated the activity of the β-1,3-glucanase (PR2) by a concentration-dependent mode at 24 h (Sanchez-Rangel et al., 2012), emphasizing the relevance of PR genes as defense signaling indicators against fungal mycotoxins.
Genes encoding for sugar efflux transporters (SWEET) were also evaluated for the first time in this study (Fig 5 e-g). The greatest expression still occurred at 48 h, especially after treatment with 5 µM FB1, where SWEET4 reached the most pronounced expression values (FC= 5.9; Fig 5 e). Several SWEET transcripts, including SWEET4, 12 and 15, accumulated in response to both the bacterium Pst and the powdery mildew fungus Golovinomyces cichoracearum and Botrytis cinerea, highlighting the potential role of these transporters in pathogen nutrition (Chen et al., 2010; Gupta et al., 2020, 2021). Previous works reported that Arabidopsis sweet11/sweet12 double mutants displayed increased resistance against the fungal hemibiotroph Colletotrichum higginsianum, both in the biotrophic and the necrotrophic colonization phase (Gebauer et al., 2017). Additionally, AtSWEET4 knockout mutants were found to be less susceptible to B. cinerea (Chong et al., 2014), suggesting that reduced carbohydrate availability correlates with susceptibility toward pathogens. Few examples in literature focusing on the role of sugar transporters in response to mycotoxins are available (Norholm et al., 2006; Vedamurthy et al., 2008; Wang et al., 2012). The expression of the hexose-specific H+-symporter SPT13 was strongly enhanced in Arabidopsis plants challenged with FB1 and the virulent (DC3000) and avirulent (AvrRPM1) P. syringae strains 2 and 4 days after the treatment, respectively (Norholm et al., 2006). Additional sugar transporters were detected upregulated by transcriptomic analysis in response to OTA (Wang et al., 2012). A further study put in evidence an altered glucose uptake and reduced sugar synthesis in sugarcane cells treated with the fungal red rot toxin produced by Colletotrichum falcatum (Vedamurthy et al., 2008).
3.4 Antioxidant compounds and enzymes involved in the ascorbate -glutathione cycle
FB1 treatment was able significantly to affect the activity of the antioxidant compounds and enzymes involved in the ASC-GSH cycle, which are generally involved in the plant defense system.
FB1 treatment had different responses according to the concentration applied. The lowest concentration of FB1, 1 µM¸ caused a marked and statistically significant decrease in ASC at all time points (Fig 6s). This same trend was observed for DHA (Fig 6b) and APX (Fig 6e). As regards DHAR, MDHAR, and GR, the activity decrease was statistically significant only at 72 h (Fig 6f, g, and h respectively), while for GSSG a decrease was observed only at 24 h (Fig 6d). Conversely, GSH markedly increased, with statistically significant differences observed at 24 h and 72 h (Fig 6c).
A different effect for almost all variables was observed when FB1 5 µM was applied. ASC levels significantly increased at 24 h and 48 h, then were comparable to the control (Fig 6a); in accordance to ASC trend, APX values were comparable (24 h and 72 h) or lower (48 h) with respect to the control (Fig 6e). DHA values did not differ from the control, apart from a statistically significant decrease at 72 h (Fig 6b). While DHAR trend fluctuated, MDHAR values remained higher than the control until 48 h and then decreased to values comparable to the control at 72 h (Fig 6f and e, respectively).
GSH and GSSG showed opposite behaviors at 24 h, with the first being significantly higher and the latter lower with respect to the control (Fig 6c and d, respectively). Then, a statistically significant decrease for GSH was only registered after 72 h. GR levels were always lower than the control throughout the assay (Fig 6h).
APX is an important H2O2 scavenging enzyme, which uses ASC as electron donor in the ascorbate-glutathione (ASC-GSH) cycle. Once oxidized to MDHA, ASC is regenerated by the GSH-dependent enzyme MDHAR. DHA, originated from the disproportionation of MDHA, can be also converted to ASC by another GSH-dependent enzyme, DHAR. Finally, GSH is regenerated by GR (Loi et al., 2020).
In both experimental conditions, FB1 affected the levels of antioxidant compounds and enzymes of the ASC-GSH cycle. When FB1 1µM was applied, the levels of the variables were generally lower, with the only exception represented by GSH. The most striking result was shown for ASC, DHA and APX, the latter being also supported by the lower levels of gene expression. These results may imply that the ASC system did not play an essential role in the H2O2 scavenging. On the other hand, we observed an increase of ascorbate at 24 and 48 h with 5 µM FB1 together with higher SWEET transcripts level, suggesting a higher availability of monosaccharides for ASC biosynthesis (Dowdle et al., 2007; Smirnoff et al., 2018; Paciolla et al., 2019).
Conversely, GSH levels were significantly higher than the control for both experimental conditions, proving that it could be actively participating in the scavenging of H2O2 also in presence of low oxidative stress. Indeed, GSH is one of the most abundant, low-molecular-weight-thiol antioxidant molecule, involved in radical scavenging and in the protection of the thiol groups of proteins and in redox signaling (Hasanuzzaman et al.,2017). The increase in GSH cannot be ascribed to an increase of GR, neither to the activity of MDHAR and DHAR. It is therefore possible that other enzymes contributed to maintain high GSH levels when FB1 was applied. GSH homeostasis is redundantly regulated at different levels, which control the synthesis ex novo, the degradation, and the regeneration from its oxidized form (Hasanuzzaman et al., 2017). Moreover, ER stress is reported to increase GSH levels in Arabidopsis, possibly due to the downregulation of GSH-dependent peroxidases (Uzilday et al., 2018)
3.5 Enzymes involved in H2O2 scavenging, H2O2 levels and lipid peroxidation
Different enzymes involved in H2O2 scavenging, namely SOD, POD, and CAT, were considered in this study to assess the effect of FB1 on the oxidative response of Arabidopsis cells.
FB1 1µM induced a slight, but statistically significant increase in SOD after 24 h and 72 h (Fig. 7a), and in POD, though only after 24h (Fig. 7b). CAT levels were also increased by FB1 1µM at 24 h; nonetheless, at 48 h and 72 h they were lower than to the control (Fig. 7c). The same trend was elicited by FB1 5µM for POD and CAT (Fig. 7B and C), while no differences with the control emerged for SOD (Fig. 7a).
H2O2 is one of the most important ROS, endowed with a relatively long half-life and high diffusion rate in water (Smirnoff and Arnaud, 2019). Due to those characteristics, at low concentrations H2O2 acts as a signal molecule, regulating the redox balance of the cell, its growth and development. Several enzymatic and non-enzymatic compounds are redundantly involved in ROS and H2O2 scavenging to assure that a physiological level is maintained.
In our experimental condition, FB1 was able to induce a rapid increase in intracellular H2O2 throughout the assay, causing reduced cell growth and, eventually, cell death.
During the first hours of exposure, intracellular H2O2 was scavenged due to an increase in CAT and POD activity, although the SOD activity increased at 24 h with FB1 1µM, contributing to H2O2 increase; this, however, together to contemporaneous increase of DAL1 and DAL2 gene transcripts kept under control the radical superoxide anion level (Basnayake et al., 2011). Later on, the system entered in physiological distress, H2O2 kept accumulating without being counteracted by CAT and POD, contributing to cell death.
Following these findings, the discrepancy in intracellular H2O2 with the results obtained for the extracellular H2O2 (which was higher with FB1 5µM) can be explained by a leakage of H2O2 from the cellular compartment to the extracellular environment. Besides, H2O2 can be produced by separate systems in the plasma membranes and cell walls, such as the NADPH-dependent oxidases which are implied in the cell wall H2O2 –dependent lignification (Habibi, 2014).
3.6. Isozymes and protein redox status
The isozyme pattern was analyzed by native-PAGE. No differences emerged between the control and the samples treated with FB1, regardless of the concentration used (data not shown). Therefore, Arabidopsis response to FB1 did not involve the induction of additional isozymes for all enzymes analyzed (APX, CAT, GR, SOD, DHAR, and POD). So far, the induction of novel isozymes with DHAR activity and involved in the defense mechanism has been reported in tomato plants for beauvericin, another Fusarium toxin (Loi et al., 2020b).
The redox state of protein-thiols appeared unchanged (data not shown), with no differences between the control and the FB1 treated samples, possibly maintained by the high GSH levels through glutathiolation, a reaction that can protect the protein thiol groups from irreversible inactivation by oxidation (Rouhier et al., 2008; 2015). The glutathiolation, that is a reversible post-translational modification consisting in a disulfide bond formation between a protein thiol and GSH, occurs more frequently in response to increased ROS (Rouhier et al., 2008).