The Mycotoxin Fumonisin B1 Induces Metabolic and Molecular Perturbations Leading to Cell Death in Arabidopsis Cell Culture

doi:10.21203/rs.3.rs-1202847/v1

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The Mycotoxin Fumonisin B1 Induces Metabolic and Molecular Perturbations Leading to Cell Death in Arabidopsis Cell Culture

https://doi.org/10.21203/rs.3.rs-1202847/v1

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Fumonisin B₁ (FB₁) is a fungal toxin produced by Fusarium spp. able to exert pleiotropic toxicity in plants. FB₁ is known to be a strong inducer of the programmed cell death (PCD); however, the exact mechanism underling the plant-toxin interactions and the molecular events that lead to PCD are still unclear. Therefore, in this work we provided a comprehensive investigation of the response of the model organism Arabidopsis thaliana at the nuclear, transcriptional, and biochemical level after the treatment with FB₁ at two different concentrations, namely 1 and 5µM during a time-course of 96 h. FB₁ induced oxidative and nitrosative bursts and a rapid cell death in Arabidopsis cell cultures, which resembled a HR-like PCD event. Different genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, resistance, and sugar transport were upregulated, especially during the late treatment time and with higher FB₁ concentration. Among the antioxidant enzymes and compounds studied, only glutathione appeared to be highly induced in both treatments, suggesting that it might be an important defense molecule induced during FB₁ exposure. Collectively, these findings highlight the complexity of the defense network of A. thaliana and provide important information for the understanding of the physiological, molecular, and biochemical responses to counteract FB₁-induced toxicity.

Agronomy

Plant Physiology and Morphology

Fumonisins

Arabidopsis

antioxidant enzymes

gene expression

cell death

reactive species

Fumonisin B₁ induces rapid programmed cell death in Arabidopsis cells, oxidative and nitrosative bursts, and differentially modulates cell death responsive genes. Glutathione is the main antioxidant involved in the defense response.

Fumonisins are toxic metabolites produced by phytophatogenic fungi belonging to the Fusarium species. Fusarium proliferatum, F. verticillioides, F. oxysporum, and F. fujikuroi are among the main producers, responsible for the contamination of several crops worldwide (Braun et al., 2018). Fusarium spp. are able to contaminate several crops of agronomic and economic relevance, such as tomato, maize, rice, and sunflower, hence representing an important health and economic concern globally (Khodaei et al., 2021). Fusarium spp. can infect plants at different stages of their development, and the production of fumonisins can be an important pathogenicity and virulence factor. Fumonisin B₁ (FB₁) is the most toxic and prevalent one Cendoya, et al., 2018), and it is classified in group 2B by the International Agency of Research on Cancer (IARC, 1993), thus resulting as a possible human carcinogen. Besides, it displays pleiotropic toxicities in animals (neurotoxicity, hepatotoxicity, and nephrotoxicity) and plants (chlorosis, necrosis, wilting, reduced growth and seed germination, and death) (Renaud et al., 2021).

Fumonisins are polyketides composed by an aminopolyol backbone structure with two tricarballylic acid side chains and an amine moiety. Their toxicity can be ascribed to their chemical structure, which resembles sphinganine, a sphingolipid with structural role in the cell membrane. In particular, the tricarballylic acid side chains and the amine moiety are the main toxic determinants (Renaud et al., 2021). At cellular level, fumonisins act as competitive substrates and inhibitors of the enzyme ceramide synthase, causing an accumulation of sphinganine and sphingosine, the natural ceramide synthase substrates, and a depletion of dihydro-sphingosine, complex sphingolipids, and ceramides in the endoplasmic reticulum (ER). This imbalance, together with the induction of oxidative stress at cytoplasm and ER level, triggers different downstream signaling pathways and epigenetic changes, eventually leading to the programmed cell death (PCD) in plants (Qin et al., 2017; Iqbal et al., 2021). Besides being structural components of the cell membrane, sphingolipids act as secondary messengers and regulate the expression of the phytohormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). FB₁ can also elicit a rapid PCD, known as hypersensitive response (HR), usually initiated by a pathogen attack and limited to the cells, which are in direct contact with the pathogen (Salguero-Linares and Coll, 2019). This response includes chromatin condensation, phenols and callose deposition, phytoalexin accumulation, rapid accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the expression of pathogenesis-related (PR) proteins (Zhang et al., 2015).

Oxidative stress also plays a pivotal role in determining FB₁ toxicity. After FB₁ exposure, increased levels of ROS, lipid peroxidation and oxidative DNA damage can be observed in vitro and in vivo due to an impairment of the redox homeostasis (Liu et al., 2019). However, the exact mechanism behind the antioxidant defense system and crosstalk among the phytohormones, sphingolipids ratio, and the resulting responses at nuclear and organelle level that led to PCD is still unclear. Likewise, it remains to be established whether the oxidative stress is caused by FB₁ or a consequence of other events that take place upon exposure (Zeng et al., 2020; Iqbal et al., 2021).

Arabidopsis thaliana is a model organism, which has been extensively used to study gene expression and toxicity mechanisms induced by FB₁ exposure at cellular level (Iqbal et al., 2019). Understanding the molecular and biochemical pathways induced by this mycotoxin is essential to counteract the toxic effects of fungal and mycotoxin contamination, and possibly to identify bioactive compounds able to minimize or neutralize them or key components that confer resistance to mycotoxin or fungal contamination. Indeed, many bioactive compounds are able to regulate these signaling pathways and to counteract the mycotoxin-induced oxidative stress (Loi et al., 2020a), and antioxidant systems to actively participate in the defense system against fungi and their mycotoxins (Loi et al., 2020b; Maschietto et al., 2016; Lanubile et al., 2015).

Therefore, the aim of this work was to provide a comprehensive investigation of A. thaliana responses upon FB₁ exposure at the nuclear, transcriptional, and biochemical level, with particular attention to the nuclear morphology, the role of antioxidant components, and the key genes contrasting cell death, in order to unravel the molecular mechanisms of the cellular defense responses induced in plants by FB₁.

2.1. Cell cultures and treatments

The Arabidopsis cell line derived from hypocotyls dissected from young plantlets of Arabidopsis thaliana (L) Heynh. Ecotype Landsberg erecta (Ler), and subcultured in liquid AT3 medium (Carimi et al., 2005). For subculture cycles, 2 mL of packed cell volume was placed in 250-mL Erlenmeyer flasks containing 50 mL of liquid medium. Cells were subcultured in fresh medium at 7-d intervals and maintained in a climate chamber on a horizontal rotary shaker (80 rpm) at 25°C with a 16 h light/8 h dark photoperiod and a light intensity of 70 µmol m⁻²s⁻¹. Treatments were performed on 3-d-old cultures. FB₁ (AppliChem, Germany) was dissolved in dimethyl sulfoxide (DMSO) at a 10 mM stock concentration. Flasks were treated with 5 µL (1 µM FB₁) or 25 µL (5 µM FB₁). Control flasks were mock treated with 25 µL DMSO, unless otherwise specified. Cells were analysed and collected 1, 3, 6, 24, 48, 72 and 96 h after treatment. Cell growth was estimated by measured the fresh weight of the cultures and vacuum filtered on a filter paper.

2.2. Cell death assessment and analysis of nuclear morphology

Cell death was estimated by Evan’s blue staining method. Evan’s blue is a dye that only stains dead cells. Briefly, 2 mL cell cultures were stained in a tube by adding 50 µL of a 0.5% w/v Evan’s blue (Sigma-Aldrich) solution. After 15 min incubation, cells were filtered through a chromatographic column (Bio-Rad, USA) and washed three times with distilled water to remove excess dye. Columns were capped and filled with 2 mL elution solution (50% v/v methanol, 1% w/v sodium dodecyl sulfate) and incubated 30 min at 55°C to promote dye release. Once filtered, the A600 of the solution was read at a spectrophotometer (Biotek, USA). In order to compensate for cell number, due to different time of culture or growth inhibition caused by treatments, the same procedure was applied to 2 mL of culture boiled for 7 min, whose A600 value was then set as 100% dead cells.

Nuclei were stained and visualized with a fluorescence microscope (DMR, Leica) as reported in De Michele et al. (2009).

2.3. ROS and RNS quantification

ROS and RNS were quantified by fluorimetric analysis using specific fluorescent dyes, as in Sharaf et al. (2019). In particular, 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA) is an intracellular marker that measures the level of oxidation inside a cell(Chen et al. 2010); dihydroethidium (DHE) is a specific marker for the superoxide anion (·O₂⁻) (Nazarewicz et al. 2013); 4-Amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-DA) is an intracellular specific marker for nitric oxide (NO)(Kojima et al. 1999); Aminophenyl fluorescein (APF) is a marker for both peroxynitrite (ONOO⁻) and hydroxyl radical (⋅OH), since the dye is unable to discriminate between the two molecules (Setsukinai et al.2003). All dyes were from Cayman Chemicals, USA, and were dissolved in DMSO. Two mL of culture was deposited in a well in a transparent 12-well polypropylene plate (Greiner, Germany) and brought to pH 7.5 by adding 20 µL 10 mM Tris buffer (final concentration 100 µM). Then, 2 µL of dye was added to the culture, with final dye concentration of 10 µM (H₂DCF-DA, DHE, APF) or 5 µM (DAF-FM-DA). Plates were incubated at 25°C for 30 min on agitation (100 rpm) in the dark. Fluorescence was measured using a Synergy H1 reader (Biotek, USA) with a bottom reader mode and gain set to 80 (H₂DCF-DA and APF) or 100 (DHE and DAF-FM-DA) and bandwidth of 9 nm. To avoid the formation of cell clumps, which affect the homogeneity of the fluorescence readout, the measurement was made in 21 different points on the well surface and averaged (“area scan” mode). Excitation and emission wavelengths for each dye were 495/525 Ex/Em for H₂DCF-DA; 495/515 Ex/Em for DAF-FM-DA and APF; for DHE, we used 405/570 nm Ex/Em instead of the commonly used 480/580 nm Ex/Em because the former setup was proved to be more selective in detecting·O₂⁻, rather than the unspecific oxidized byproduct 2-OH-ethidium.

Extracellular H₂O₂ was measured by using the xylenol orange method. Briefly, 2 mL of culture was filtered through a chromatographic column (Poly-Prep; Bio-Rad, USA) to separate cells from the growth medium. An aliquot of 500 mL of the flow through was added to an equal volume of assay reagent (500 mM ferrous ammonium sulfate, 50 mM H₂SO₄, 200 mM xylenol orange, and 200 mM sorbitol) and incubated for 45 min in the dark. The H₂O₂-mediated oxidation of Fe²⁺ to Fe³⁺ was determined by measuring the A₅₆₀ of the Fe³⁺⁻xylenol orange complex. All reactions were carried out at least in duplicate, and their reproducibility was checked.

Intracellular H₂O₂ was determined by fluorescence using dihydrorhodamine 123 (DHR123; Sigma-Aldrich, St. Louis, MO) (Qin et al. 2008). Briefly, an aliquot of frozen cell culture (0.5 g) was incubated for 15 min with 10 µL of a solution containing sucrose (30% w/v) and DHR123 115 µM. Green fluorescence related to intracellular H₂O₂ was observed in a fluorescent microscope (DMLS, Leica) with an excitation filter of 450–490 nm and a barrier filter of 510 nm

2.4. Gene expression and DNA analysis

Genomic DNA was extracted by grinding cells with liquid nitrogen, followed by the Doyle and Doyle method (Carimi et al., 2004) and quantified at a spectrophotometer. For DNA fragmentation analysis, 10 µg of each sample was electrophoresed on 1% (w/v) agarose gels containing 1× TAE (40mM Tris–acetate, 1mM EDTA) and stained with ethidium bromide.

Expression of the senescence marker SAG12 was performed as in (Carimi et al., 2004), by using 18S as an internal standard (Ambion, USA). Cells from 14 d old culture were used as positive control for SAG12 expression (Carimi et al 2004).

For real-time reverse transcription-PCR (RT-PCR) gene expression, Arabidopsis cells were ground under liquid nitrogen with a pestle and mortar, and total RNA extraction and purification were carried out based on Lanubile et al. (2013, 2015). Real-time experiments were performed on cells collected at 24 and 48 h after treatment with 1 and 5 µM FB₁ solution using the FluoCycle^TM II SYBR Green master mix (EuroClone S.p.a., Milan, Italy) and the CFX-96 device (Bio-Rad, Hercules, CA, U.S.). One µg of total RNA was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc. Waltham, Massachusetts, U.S.). Twenty ng of single strand cDNA determined by fluorometric assay (Qubit, Thermo Fisher Scientific) were used for real-time RT-PCR. Relative RT-PCR was performed under the following conditions: 95°C for 3 min and 40 cycles at 95°C 15 s, 55-60°C for 30 s, followed by a melting curve analysis (Lanubile et al., 2013, 2015). Three technical replicates (within each biological replicate) were employed for each tested sample and template-free negative controls. Gene-specific primers are listed in Supplemental Table S1. Relative quantification was normalized to the reference housekeeping gene Actin2. Fold changes (FC) values in gene expression were calculated using the 2^−ΔΔCt method (Schmittgen and Livak, 2008) and calibrated on the mock-treated cells.

2.5. Determination of Proteins Extraction and Quantification

One gram of cell culture was harvested by filtration as described above and grounded with liquid nitrogen in a porcelain mortar. Then, the extraction buffer consisting of Tris-HCl 50 mM pH 7.8 0.05% w/v cysteine and 0.1% w/v bovine albumin was added in a ratio 1:2 w/v. The homogenate was centrifuged at 1,000 × g for 5 min. The supernatant was re-centrifuged at 25,000 × g for 20 min and the resulting supernatant was desalted by dialysis against 50 mM Tris-HCl pH 7.8, and used for enzyme activity measurements and for the electrophoretic analyses. All procedures were carried out at 4°C. The protein content was quantified with a Protein Assay kit from Bio-Rad (Hercules, CA, USA) with bovine serum albumin as the standard.

2.6. Enzyme Activity Measurements

Enzyme activities were determined spectrophotometrically, by monitoring the rate of substrate oxidation or product formation at specific wavelengths. In particular, APX (EC 1.11.1.11), CAT (EC 1.11.1.6), POD (EC 1.11.1.7), SOD (EC 1.15.11), MDHAR (EC 1.6.5.4), and GR (EC 1.6.4.2) were tested according to Paciolla et al. (2008) and Mastropasqua et al. (2012). DHAR (EC 1.8.5.1) was determined according to Loi et al. (2020).

2.7. Ascorbate and glutathione pools

For ascorbate and glutathione determinations, 0.5 g cells were packed in 2 mL tubes, resuspended in 1.5 mL of a 5% w/v metaphosphoric acid solution and frozen in liquid nitrogen.

Ascorbate and glutathione pools were determined according to Loi et al. (2019).

2.8. Lipid peroxidation

Lipid peroxidation was monitored by measuring the end product malondialdehyde (MDA). Briefly, 0.7 g of Arabidopsis cells were grounded in a porcelain mortar with liquid nitrogen and dissolved with 0.1% trichloroacetic acid with a ratio of 1:4 (w/v). After centrifugation at 12,000 × g for 10 min, the supernatant was diluted 1:1 with a solution containing 20% trichloroacetic acid and 0.5% thiobarbituric acid (TBA) and incubated for 30 min at 90°C. The reaction was stopped in ice and the samples centrifuged at 12,000 × g for 10 min. The resulting supernatant was used for the determination of MDA-TBA complex by spectrophotometric measurement at 532 nm (extinction coefficient 155 mM⁻¹ cm⁻¹).

2.9. Electrophoretic analyses

Native-PAGE

Native-PAGE was performed on PAGE (7.5% T; 4.0% C). Fourty µg of total proteins were loaded in each lane. The electrophoresis was performed in a Mini Protean System (Bio-Rad, Segrate, Italy) filled with running buffer (25 mM Tris and 1.9 M glycine). The run was performed at 32 mA for 3h. After the electrophoretic run, gels were used for activity staining for the different enzymes. Activity staining was performed by incubating the gels in specific buffers. APX, CAT, GR were detected as described by Paciolla and colleagues (2016), DHAR according to Loi et al. (2020) while SOD by Villani and colleagues (2021), For POD activity staining, the gel was incubated in Tris-acetate 0.1 M, pH 5 containing H₂O₂ 0.32 mM and 1-methoxynaphthol 1mM. After incubation at 27°C for 15 min, POD appeared as blue bands on a light background.

Protein thiol labelling

Protein SH groups were labelled with the fluorescent probe monobromobimane (mBBr) according to procedure reported by Gobin et al. (1997). 150 µg of total proteins were loaded on sodium dodecyl sulphate (SDS) gel (10% T, 4% C). SDS-PAGE was performed according to Laemmli (1970). The protein content was assayed with the Bio-Rad kit. The electrophoretic run was performed as previously described for Native-PAGE. After the run, the proteins was fixed with trichloroacetic acid 12% (w/v) for 1 h and then the gel was incubated in a solution consisting of 40% methanol, 10% acetic acid, and 50% water for 10 h to remove excess mBBr. The fluorescence of thiol-bound mBBr was detected by placing the gel on a UV-transilluminator (365 nm). The resulting fluorescence emission is indicative of the thiol presence in the analyzed proteins (Paciolla et al., 2001). The intensity of fluorescent bands was analysed with UTHSCA Image Tool software.

2.10. Statistical analyses

At least three biological replicates were performed for all experiments. For cell growth, mortality, ROS and RNS analysis means and standard deviations are presented. Variance among replicates was first tested by F-test, to check for equal or unequal distribution. Then, treatments were compared for significant difference at p = 0.5 by T-test.

For gene expression and other parameters analyzed, standard deviations of the means were calculated on three biological replicates. One-factor analysis of variance (ANOVA), followed by Tukey’s HSD test (p < 0.05), was performed on the observed means of FC gene expression other studied parameters values to set significant differences between times of treatment (24 and 48 h for gene expression; 24, 48 and 72 h for enzyme activity, ascorbate, glutathione and lipid peroxidation) within each FB₁ concentration and between FB₁ concentrations (1 and 5 µM) within each time of treatment.

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 FB₁1µM 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 FB₁5µM and at 72 h for FB₁1µM, 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 FB₁exposure (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 FB₁exposure, 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 FB₁and 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 FB₁treatment 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 FB₁exhibited 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 FB₁5 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 H₂O₂scavenging 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 FB₁1µM 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 H₂O₂scavenging. On the other hand, we observed an increase of ascorbate at 24 and 48 h with 5 µM FB₁together 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.

FB₁1µM 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 FB₁1µM 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 FB₁5µM for POD and CAT (Fig. 7B and C), while no differences with the control emerged for SOD (Fig. 7a).

H₂O₂is 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 H₂O₂scavenging 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 FB₁1µM, contributing to H₂O₂increase; 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 FB₁5µM) can be explained by a leakage of H₂O₂from the cellular compartment to the extracellular environment. Besides, H₂O₂can 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).

FB₁ exposure to Arabidopsis cell culture induced metabolic and molecular perturbations that lead to cell death due to a strong oxidative and nitrosative damage. The transcriptional analysis revealed that FB₁ was able to induce different genes involved in the regulation of PCD, antioxidant metabolism, photosynthesis, resistance and sugar transport. Among the biochemical parameters studied, GSH seemed to be the main antioxidant compound involved in the defense response to the fumonisin exposure, highlighting the pivotal role of this multifaceted antioxidant molecule.

Collectively, the outcomes of this work showed that FB₁ exposure induced several, redundantly regulated defense networks in Arabidopsis cells, pointing out the complex and dynamic plant-toxin interaction. Although further studies are needed to completely elucidate this multifaceted defense network, the results of this work lay the basis for a broad comprehension the mycotoxin-plant interaction at the physiological, molecular, and biochemical levels.

Funding

This work was financially supported by PRIN 20094CEKT4 of the MIUR, Italy and by H2020‐E.U.3.2-678781-MycoKey-Integrated and innovative key actions for mycotoxin management in the food and feed chain

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contributions

Costantino Paciolla, Alessandra Lanubile and Roberto De Michele contributed to the study conception and design. Costantino Paciolla, Alessandra Lanubile, Roberto De Michele and Safieh Fakhari prepared the material, collected data and performed the experiments. Alessandra Lanubile, Roberto De Michele, Martina Loi and Costantino Paciolla analyzed the data. Costantino Paciolla, Alessandra Lanubile, Roberto De Michele, Martina Loi and Adriano Marocco wrote and reviewed the manuscript draft. Adriano Marocco and Costantino Paciolla supervised the work and provided the funding. All authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Silvana De Leonardis for her technical support

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The Mycotoxin Fumonisin B1 Induces Metabolic and Molecular Perturbations Leading to Cell Death in Arabidopsis Cell Culture

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Abstract

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Key Message

1. Introduction

2. Materials And Methods

2.1. Cell cultures and treatments

2.2. Cell death assessment and analysis of nuclear morphology

2.3. ROS and RNS quantification

2.4. Gene expression and DNA analysis

2.5. Determination of Proteins Extraction and Quantification

2.6. Enzyme Activity Measurements

2.7. Ascorbate and glutathione pools

2.8. Lipid peroxidation

2.9. Electrophoretic analyses

Native-PAGE

Protein thiol labelling

2.10. Statistical analyses

3. Results

Conclusions

Declarations

References

Supplementary Files

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