Sulfite Oxidase Activity Level Determines Sulfite Toxicity Effect in Leaves and Fruits of Tomato Plants

Plant sulfite oxidase )SO( is a molybdo-enzyme responsible for the oxidation of excess SO 2 /sulfite into non-toxic sulfate. The effect of toxic sulfite level on leaves and fruits was studied in tomato plants with different SO expression levels: wild-type (WT), SO overexpression (OE) and SO RNA interference (Ri) plants. Leaf discs and ripe fruit of plants lacking SO were more susceptible, whereas SO OE plants were more resistant as revealed by remaining chlorophyll content and tissue damage levels. Application of molybdenum further enhanced the tolerance of leaf discs to sulfite by enhancing SO activity in SO OE lines, but not in WT or Ri plants. Notably, incubation with tungsten, the molybdenum antagonist, overturned the effect of molybdenum spray in SO OE plants, revealed by remaining chlorophyll content and SO activity. The results indicate that SO determines, in tomato leaves and ripe fruits, the resistance to toxic sulfite and the application of molybdenum enhances sulfite resistance in OE plants by increasing SO activity. The results suggest that overexpressing SO mechanism can be employed in agriculture with or without molybdenum application, for the development of more tolerate crops and vegetables to higher concentrations of sulfite/SO 2 containing postharvest treatments.


Introduction
Sulfur is the least abundant macronutrient in plants, comprising approximately 0.1% of the dry matter. As a component of a molecule it is mostly involved in catalytic or electrochemical functions rather than as a structural component such as are nitrogen and carbon [1]. Sulfur is found in amino acids (cysteine and methionine), oligopeptides (such as glutathione and phytochelatins), vitamins and cofactors (such as biotin, thiamine, CoA and S-adenosyl methionine), and in a variety of secondary products such as glucosinolates [2].
Sulfate (SO4 2-) is the primary source of sulfur in plants [3,4]. Sulfate is transported from soil into roots where it can remain or be distributed [5]. The reduction of sulfate by the sulfate reduction pathway into cysteine initiates by its adenylation catalyzed by ATP sulfurylase (ATPS) in both chloroplast and cytosol. The resulting adenosine 5-phosphosulfate (APS) forms a branching point in the pathway as acted by different enzymes [4,6]. In the primary sulfate assimilation, APS is first reduced by APS reductase (APR) to sulfite, which is further reduced to sulfide by the chloroplast-localized, ferredoxin dependent sulfite reductase (SiR) [7,8]. The O-Acetyl-L-serine (OAS) synthesized from serine and acetyl-Coenzyme A catalyzed by serine acetyltransferase together with sulfide is catalyzed into cysteine by OAS (thiol) lyase (OASTL) [9]. Sulfite generated by APR or obtained from atmospheric SO2 is a highly toxic intermediate. A bulk of sulfite is normally channeled in the assimilatory reduction pathway to generate sulfide catalyzed by sulfite reductase (SiR), yet excess sulfite is be oxidized effectively back to sulfate by the peroxisome localized sulfite oxidase (SO), acting as the safety valve in the sulfate reduction pathway [10]. SO is catalyzing the reaction in which two electrons are 4 transferred from sulfite to the molybdenum cofactor (Moco) redox center, which are subsequently transferred to molecular oxygen with simultaneous production of hydrogen peroxide (H2O2) and sulfate [11]. The generated H2O2 can further oxidize another sulfite molecule to sulfate nonenzymatically or acted by the peroxiosomal catalase upon low cellular sulfite concentration to H2O and O2 [12].
SO2 is a gaseous pollutant. It is a major atmospheric contaminant resulting from the combustion of sulfur-rich fossil fuels and from natural sources such as microbial activities, forest fire and volcanic eruptions. SO2 enter plants via their stomata and readily hydrates to form the sulfite ions, HSO3and SO3 2- [13]. At sub-toxic levels, plants are able to utilize SO2. Indeed, sulfur assimilation and biomass production are reported to be positively correlated with SO2 in the air [14]. Yet, above a certain threshold, sulfite toxicity leads to visible symptoms of chlorosis and necrosis causing reduction in plant growth which leads to severe loss in yield [15]. Sulfite anions (HSO3and SO3 2-) are nucleophilic agents that are able to attack DNA, proteins and lipids and thus affect plant growth and vitality [16]. Sulfites can interfere the thiol/disulphide functional groups and disrupt the regulation of key metabolic processes, including photosynthesis and respiration [17,18], by inactivation of proteins like thioredoxins [18]. Sulfite can degrade lipids by oxidation, which leads to lipid peroxidation, resulting in cell membrane dysfunction and damage [16]. The susceptibility to SO2 differs between the plant species in combination with the duration and concentration of SO2 in the atmosphere [12,19].
By the use of tomato wild type, two SO overexpression (OE) and two SO RNA interference (Ri) independent lines it was shown in this study that SO level determines the resistance to toxic sulfite in tomato leaves and ripe fruits and the 5 application of molybdenum (Mo) further enhances the sulfite resistance in OE tomato plants by increasing SO activity. The results suggest that the overexpressing SO mechanism can be used in agriculture for the development of SO transformed lines in crops and vegetables, which can tolerate higher concentrations of pesticides or sulfur containing postharvest treatments of fruits and vegetables; and application of Mo on them for better results.

Chlorophyll content and SO activity
The effect of SO expression levels of the plants on the capacity to detoxify toxic levels of sulfite was investigated in wild type and SO modified tomato lines. Leaf discs were treated with 7 mM sodium sulfite for 24 hours. Chlorophyll content, a sensitive indicator of leaf health was monitored on the leaf discs after treatment.
SO enzyme activity was determined in the crude protein extracted from leaves of wild type and SO modified plants.
Leaf discs from the first fully developed leaf of wild type (RR), SO OE (OE 13-6/6 and OE 12-5/7) and SO Ri (Ri 421 and Ri 131) were taken for the experiment. No damage of the leaf discs was observed in the control treatment ( Fig. 1A), while symptoms of chlorosis and damage from the periphery were observed in the leaf discs incubated with 7 mM Na2SO3 (Fig. 1B). The least damage occurred in the leaf discs of both the OE lines, followed by the wild type and Ri lines. In wild type, the observed damage was intermediate, while Ri lines were strongly affected by the sulfite treatment. The results show that the tomato lines with higher SO expression levels were less affected by the sulfite treatment as compared to the lines with lower SO expression levels (Fig. 1B).

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Additionally, remaining chlorophyll content was measured in the leaf discs 24 hours after sulfite treatment. A higher amount of remaining chlorophyll was found in OE lines, whereas it was lower in Ri lines as compared to the wild type ( Fig. 2). Remaining chlorophyll content in RR line was 60.7% of the control. In SO OE lines, remaining chlorophyll content in OE 13-6/6 and OE 12-5/7 was 22.4% and 32.5%, respectively, higher as compared to the wild type. While in SO Ri lines, the remaining chlorophyll content was reduced to 51.5% and 55.9% for line Ri 421 and Ri 131, respectively, compared to the wild type (Fig.   2). The results show the remaining chlorophyll content in the leaf discs after sulfite treatment was higher for the tomato lines having higher SO expression levels and vice versa.

Effect of molybdenum and tungsten application on wild type and SO modified plants
The The strongest damage after sulfite treatment was found in H2WO4 (W) incubated leaf discs followed by DDW incubated discs and less symptoms of damage and chlorosis were observed in Na2MoO4 (Mo) incubated discs (Fig. 7).
In all DDW, Mo or W incubation treatments, Ri 421 line was more affected by the sulfite treatment, which led to symptoms of chlorosis and damage on the discs, while OE 13-6/6 line was least damaged as compared to RR and Ri line.  7).  Additionally, the effect of DDW, Mo and W pretreatment on SO enzyme activity was studied in wild type and SO modified plants. SO enzyme activity was found to be highest in the OE 13-6/6 line followed by RR line and Ri 421 line respectively (Fig. 9). Mo pretreatment significantly increased SO enzyme activity in the OE 13-6/6 line while no effect was observed in leaves pretreated with W. In the RR line, an increasing, but not significant, tendency in SO enzyme activity was observed in Mo treated leaves. In the Ri 421 line no effect of Mo and W pretreatments was observed on SO enzyme activity (Fig. 9).

Effect of sulfite application on ripe fruits
The effect of sulfite on ripe fruits was observed in wild type and SO-modified  The sulfite content in the pericarp was analyzed following the dipping treatment.
Wild type and both OE lines did not differ significantly after sulfite treatment as compared to their water dipped counterparts (Fig. 11), whereas, SO Ri lines displayed a significant augmentation in pericarp sulfite levels after sulfite dipping.

Discussion and Conclusions
SO in plants is a molybdenum cofactor containing enzyme. The SO gene was thought to possess a housekeeping function that was revealed by its basic expression levels in Arabidopsis plant organs [10]. In addition, plant SO is thought to play a role in the cellular sulfur turnover [12,17] or in protecting the thioredoxin system from damage [18]. It is further speculated that SO plays a role in stress signaling, since it has the ability to produce H2O2, as a reaction product [12]. However, when plants are exposed to elevated SO2 conditions, SO is the major enzyme to protect the plants against SO2 toxicity by oxidizing sulfite to sulfate [10,20]. Moreover, it has been shown recently that SO in coregulation with APR plays a role in driving an internal sulfate-sulfite cycle for fine tuning of sulfur flux in the plants [21]. Nevertheless, overexpressing APR, an enzyme in the sulfur assimilation pathway, resulted in adverse effects in the transgenic plants including chlorosis and inhibition of growth [22]. In this study Sulfite is a toxic metabolite that is formed during the sulfur reduction pathway [7].
Sulfite thus formed is normally reduced in the sulfur assimilation pathway catalyzed by sulfite reductase in the chloroplast to form sulfide, which is then incorporated into sulfur containing amino acids [7]. Contrasting to this pathway, sulfite can be oxidized to sulfate by the molybdenum cofactor containing enzyme SO [12]. Here, we showed that the leaf discs from plants lacking SO (Ri lines) were more susceptible to the externally applied sulfite, showing the symptoms of chlorophyll degradation and damage from the periphery (Fig. 1).
Moreover, the leaf discs of SO OE lines showed the least damage. The wild type, where SO expression was not altered was more damaged than the OE lines and less than Ri lines (Fig. 1). Remaining chlorophyll content, a sensitive indicator of leaf health, supports this result, where it is 27% higher in OE lines and 54% lower in Ri lines as compared to wild type (Fig. 2). This resistance to the sulfite in OE lines may be provided by a higher SO activity level and the susceptibility of Ri lines to sulfite is due to lack of SO. These results are supported by a 2 fold increase in SO activity in OE lines compared to wild type (Fig. 3). The Ri lines, nevertheless, did demonstrate some SO activity, which is likely to be attributed to non-specific activity. Such activity has been reported in the non-leaky molybdenum co-factor mutants of Nicotiana plumbaginifolia, which should have no SO activity [11]. These results support the findings of Brychkova et al. [10] and Lang et al. [20] who showed a major role of plant SO for protecting plants against SO2/sulfite toxicity. Thus, plant SO is the major enzyme to act on the externally applied sulfite and its level in the plants determines the level of toxicity in plants.
Mo is an essential micronutrient in plants and animals. The requirement of Mo for plant growth was first reported by Arnon and Stout [23] using hydroponically grown tomato. It itself is not biologically active, but is rather predominantly found to be an integral part of the organic pterin complex called molybdenum cofactor [24]. These molybdoenzymes catalyze important transformations in the global sulfur, nitrogen and carbon cycles [25]. Leaf discs of the Mo sprayed OE 13-6/6 plants in our experiment were more tolerant to sulfite treatment with less damage, retaining more chlorophyll content compared to non sprayed (Figs. 4 and 5). Indeed, foliar application of Mo on Mo-deficient plants effectively rescued the activity of the molybdoenzyme NR in grapes [26]. Moreover, application of molybdate in the nutrition medium or as a foliar spray increased the yield accumulation of the seawater grown halophyte Salicornia europaea by enhancing the activity of the molybdoenzymes NR and XDH [27]. Therefore, we determined the SO activity in Mo sprayed plants. As expected, a significant increase in SO activity was achieved in OE 13-6/6 line after Mo application as compared to non-sprayed, but no effect was observed in wild type and Ri 421 line (Fig.6). We further investigated the effect of sulfite pretreatment on SO activity. Incubation of leaves in 3 mM Na2SO3 for 8 hours revealed increased activity in Mo sprayed OE 13-6/6 leaves and not in wild type or Ri 421 line.
However, after sulfite pretreatment, no further increase in SO activity was found in wild type and SO modified lines (Fig. 6). Similar results have been reported by Brychkova et al. [10], where SO protein and transcript levels were not highly sensitive to SO2 exposure, rather, SiR transcripts were highly induced in a SO  [29]. For example, an experiment performed in rats that were fed with tungstate or molybdate followed by subsequent measurement of the metal content in sulfite oxidase and xanthine oxidase, revealed that both metals could be incorporated into pterin cofactor of the enzymes, but the tungsten containing enzymes were completely inactive [30]. reported in barley roots with the Mo application in the nutrient medium [31].

Incubation of leaf cuttings for 24 hours in
However, this effect was not observed in W pretreatment suggesting a Mo specific increase in SO activity in the OE line (Fig. 9). Moreover, the leaf pretreated with W resulted in no differences for the remaining chlorophyll content between wild type, OE and Ri lines of W pre-treated leaves (Figs. 7 and 8). This might reflect the W toxicity in plants. It has been reported that W treatment in barley for 9 days resulted in decreased activity of molybdoenzymes AO and XDH in the leaves with reduction in growth [31]. Thus, pretreatment of Mo significantly increased the SO activity in SO OE line, which however was not achieved by W. In the latter case, W might be bound to the metal binding site in molybdenum cofactor resulting in a non-functional cofactor, which cannot participate in sulfite reduction. Since the W incubation time in our experiment was limited to 24 hours, the cofactor which already bound Mo might not be shifted by W, resulting in similar SO activity as in the control treatment (DDW) (Fig. 9).
SO2/SO3 has been used extensively in post-harvest treatment of the fruits and vegetables in order to keep the quality such as to prevent browning or to disinfect the products [32]. SO2 fumigation was shown to be very effective for prevention of fungal decay such as Botrytis cinerea in grape berries and blueberries [33,34]. However, the effectiveness of SO2 was strongly depend on concentration and timing of exposure [35]. Thus, the toxicity of the SO2/SO3 for both, plants and humans, is considered as a limiting factor of the usage of SO2.
Here, we suggest that the sulfite treatment of fruits can be effectively used for the treatment of the fruits of OE lines or species with naturally high expression Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 March 2020 Peer-reviewed version available at Agronomy 2020, 10, 694; doi:10.3390/agronomy10050694 of SO (Fig. 10). Moreover, effective sulfite utilization in the fruits of OE line would allow balancing the sulfite level in treated fruits (Fig. 11).  Germination of seeds was carried out in Petri dishes lined with wet filter paper at room temperature in the dark. The seedlings were transferred to soil in pots

Chlorophyll content determination
Chlorophyll content was determined by extracting the leaf disc samples in 80% ethanol for 48 h at 4° C in the dark. The extract was centrifuged at 12,000 g for 10 minutes. The resulting supernatant was diluted 10 times in 80% ethanol and the color was measured as the absorbance at 652 nm using a UV/VIS spectrophotometer (JASCO, model V-530). Total chlorophyll content was calculated as described by Arnon [36] and remaining chlorophyll content was expressed as % of control.

Protein extraction from the leaf samples
Crude proteins were extracted as done before [37]

Determination of protein concentration
Total soluble protein content was estimated by a modified Bradford procedure, using crystalline bovine serum albumin as a reference [38]. Briefly, protein extracts from different lines of tomato plants were diluted at a ratio of 1:25 with DDW and mixed with Protein Assay at a ratio of 1:10. The resulting color was measured as the absorbance at 595 nm in UV/VIS spectrophotometer (JASCO, model V-530) and the protein concentration was calculated.

Assessment of SO enzyme activity
SO enzyme activity was determined in the crude protein extracts of the leaf.
The activity was determined using 2 or 10 µg of soluble protein. The Fuchsine color reagent was composed of a fresh mixture of reagent A, B and DDW at a ratio of 1:1:7. Reagent A was a 0.04% Pararosaniline solution discolored in 2.3 M H2SO4 and reagent B was composed of 3.2% formaldehyde.
The reaction was started by adding soluble protein to 0.1 mM freshly prepared Na2SO3 and then incubated for 5 minutes at 30º C. The reaction was terminated by adding the color reagent into the reaction mixture. Another set of samples were immediately stopped after the addition of the substrate (Na2SO3) with the color reagent. The absorbance of the resulting color was measured at 540 nm in a spectrophotometer (Sunrise, Tecan, Pharmatec instrumentation ltd, Israel).
The readings were compared with the known standard of freshly prepared Na2SO3. The final SO activity was expressed in µmol SO3 per mg protein per minute. Ripe fruits from wild type and SO modified plants were harvested with intact calyx. Similar fruits were selected in respect to the color, size and calyx freshness. The fruits were dipped completely in the treatment solution containing 0 or 200 mM Na2SO3 for two hours. After removal from the treatment solution, the fruits were wiped gently with blotting paper to remove the excess solution. Subsequently, the fruits were kept at room temperature for 24 hours.

Sulfite treatment of fruits
Appearing symptoms of the sulfite treatment were observed on the fruits and photographed (Nikon Coolpix-4500) after removing the calyx for precise symptom observation. Additionally, 200 mg fruit samples were taken from the pericarp, snap frozen in liquid Nitrogen and stored at -80º C for further examination. Sulfite level in the tomato fruits was determined as described above. Briefly, tomato fruit samples were extracted with DDW in the ratio of 1:4 (w/v). The resulting extract was centrifuged for 15 minutes at 12,000 g. The supernatant was collected and kept on ice. The sulfite content was determined colorimetrically by using the Fuchsin color reagent as described above. The color reagent was added to the plant extract in the ratio of 1:3 and the resulting color was measured after 10 minutes at 540 nM in a spectrophotometer (Sunrise, Tecan. Pharmatec Instrumentation ltd, Israel). Sulfite content was determined against a known a standard solution of Na2SO3 and expressed in nmol per g fresh weight.

Statistical analysis
Significant differences between treatments were analyzed by appropriate single or multi-factorial analysis of variance (ANOVA) using the JumpIn 5.0.1a software package. When ANOVA indicated significance, multiple comparison of