Soil Ni concentrations
The peat-sand mix (1:2 v/v) used as growing substrate was analysed by XRF and ICP-MS to determine the Ni concentration at the starting condition. The results evidenced that the Ni mean content of the substrate was 32 mg kg-1 (range 31-32 mg kg-1). This mean Ni content was assumed as the background value for the whole experiment. The separate analyses of the two components (peat and sand) evidenced that only the sandy fraction of the mixture was characterized by Ni content above the instrumental detection limits (range 46-47 mg kg-1) thus representing the only component of the substrate mix to contribute significantly to the initial Ni content of the substrate mix.
Considering the relative nickel loss, at the end of the experiment a significant amount of Ni, added as NiSO4*6H2O, was leached from soil. Relative Ni output increases in a linear way (Figure 1), from Ni 30 to Ni 300. Nevertheless, the final Ni concentration in soil resulted always higher than the starting value of the untreated substrate.
Plant biomass development and fruit production in response to Ni
Considering plant productivity in terms of fruit produced (red, green, and total), biomass and fruit Ni accumulation, Spearman’s correlations rank (Table 1) does not highlight significant differences between Ni concentrations in fruits and fruit biomass or number.
The Ni treatments do not correlate with tomato productivity while cultivars have negative significant correlations respect to the total number of fruits produced (ρ = -0.36 p<0.05; Table 2), but not respect to the fruit biomass. Green (G, unripe) and red (R, ripe) fruits do not show significant correlations respect to the other parameters.
Since cv ‘Standard’ is more productive respect to cv ‘Ingrid’ in terms of fruits produced, productivity data were analysed grouped per cultivars obtaining the same results previously cited.
The Kolmogorov-Smirnov two-sample test between controls and Ni treatments for Ni concentrations in fruit and fruit biomass (Table 3) showed that there is a significant difference between Ni in red, green and total fruit starting from Ni 60 only for cv ‘Standard’ and Ni 30 for cv ‘Ingrid’. However, there are no significant differences for fruit biomass and productivity between controls and Ni treatments.
In addition, the Kolmogorov-Smirnov two-sample test to evaluate significant differences between controls and Ni treatments respect to plant biomass (root, stem, leaf, fruit DW and dry matter DM) (Table 4) revealed significant difference for ‘Standard’ from Ni 60 for stem (DW and DM), and from Ni 300 for leaf (DM). For ‘Ingrid’ revealed significant difference from Ni 60 and Ni 120 for stem DM, and from Ni 300 for leaf (DW and DM).
Summarizing, plant biomasses show significant differences in response to increasing Ni levels with a clear evidence at Ni 300.
The same test between the two cultivars revealed significant differences between fruit and leaf DM, higher in ‘Standard’, supporting the evidence of a higher productivity of ‘Standard’ respect to ‘Ingrid’ (Table 5).
Ni accumulation in tomato fruit
The one-way ANOVA performed on Ni treatments respect to Ni concentrations in soil and tomato at the end of the experiment (Figure 2) highlighted significant differences from Ni 120 (P=0.0002) and Ni 300 (P=0.0001) respect to the control for soils and tomatoes with a marked significant difference also from Ni in tomatoes between Ni 120 and Ni 300 (P= 0.0001).
The Pearson’s correlation between final concentrations of Ni in soil respect to Ni in tomatoes is highly significant (r=0.83 P<0.01).
Tomato allergens expressed under Ni stress
Protein extracts preparation from tomato samples
Figure 3 shows the protein concentration in the total extracts, ranging from 0.07 to 0.51 mg g-1 of the fruit. The sample showing the highest concentration is C-’Ingrid’-G, followed by C-’Standard’-G and Ni 60-’Ingrid’-R. The Figure shows that the protein concentration found in both the controls of green tomato (C ‘Standard’-G and C ‘Ingrid’-G) is higher than that observed in the same samples after Ni-treatments.
Analysis of LTP and TLP content in tomato protein fractions using biochemical methods
The analysis of RP-HPLC profile of tomato total extracts showed that the LTP detection and the estimation of its amount in the samples was not easy. This was especially due to the low concentration of this allergen compared to the other protein components. To overcome this issue, considering that LTP proteins are characterized by a basic isoelectric point, a fraction enriched in basic proteins was obtained from total extracts by separations with an anion exchanger resin. The samples were then concentrated as reported in the Materials and Methods section and their protein profile was obtained by RP-HPLC. As an example, Figure 4 shows the RP-HPLC profile of the fraction containing the basic proteins obtained from the extract of C ‘Standard’-R sample. The eluted peaks were collected and analysed by direct amino acid sequencing. Peaks eluted at 34.2 min and 34.8 min both provided the same N-terminal sequence, LSCGQVT. The similarity search against the UniProtKB database, with the BLASTP algorithm on the ExPASy server, allowed the identification of both the peaks as 9 kDa LTP, Sola l 3. At least two 9k-LTP found in the UniProtKB database had the experimentally obtained N-terminal sequence (accession numbers A0A3Q7HZ96 and K4D1U9). They have been labelled as Sola l 3a and Sola l 3b (Figure 4). Therefore, the detection in the RP-HPLC elution profile of more than one LTP peak indicates the presence of isoforms in the analysed samples. Figure 5 shows the amount of Sola l 3 estimated in the analysed tomato samples. It can be observed a certain variability of the Sola l 3 isoforms. However, it is not possible to observe any correlation between LTP concentration and the concentration of Ni applied in the treatments.
The component eluted at 49.2 min was identified as TLP (Sola l TLP) by N-terminal amino acid sequencing that provided the following sequence ATKEVRNNCP (Accession number in UniProtKb P12670). Figure 6 shows a decrease of TLP in the standard cultivar as a function of the increasing Ni concentration. The same effect is not observed in the ‘Ingrid’ cultivar.
Analysis of allergens content in the tomato samples by IgE inhibition tests
The allergens contained in the samples treated with Ni 300 were investigated by immunological tests and the results were compared with those of controls made of untreated tomato. Two samples of Ni 300-treated red tomato and green tomato were analysed with the SPHIAa method36 on the FABER system37. Figure 7 shows the IgE-binding inhibition results recorded on some allergens spotted on the FABER biochip, namely the tomato fruit extract, tomato seed extract, Bet v 1 and a Bet v 1-like protein, three profilins and seven LTP (see Table 6 for details). In line with the observation that these tomato samples contained a very low number of seeds, results obtained show that the inhibition on the entire fruit extract is high, whereas lower values were recorded for the tomato seed extract.
The presence in the fractions of both, red and green fruits, of tomato 7k-LTP, Sola l 6, was indicated by the IgE-binding inhibition (100%) recorded on this allergen spotted on the FABER biochip. Inhibition with variable values was also observed on all the seven 9k-LTP contained in the biochip. Nevertheless, the inhibitions produced by red tomato on these LTP appear independent of Ni treatment. Differently, compared with the control, results obtained with Ni-treated green tomato show a higher inhibition on the peach LTP, Pru p 3, and to a lower extent on other LTP, such as the peanut Ara h 9, the kiwifruit Act d 10 and the maize Zea m 14. All together, these results suggest a higher concentration of LTP Sola l 3 in Ni 300-treated green tomato compared to the untreated samples.
Ni-treated tomato inhibited Bet v 1 with a lower efficiency, compared to the untreated samples. This result suggest that nickel could induce a decrease of a tomato Bet v 1-like allergen. However, the result obtained on the Bet v 1-like allergen Mal d 1 did not produce the same result. Compared to the untreated tomato, both red and green Ni 300-treated ones showed a significant lower IgE-binding inhibition on the three tested profilins. This result is consistent with a reduction of profilin concentration in Ni-treated tomato. In addition to LTP, profilin and Bet v 1-like allergen, IgE-binding inhibition was detected also on the pomegranate GRP. Therefore, GRP may represent a new potential tomato allergen. The Ni-treatment seems to give only a weak effect on the GRP concentration in tomato.
Proteins response to Ni in tomato samples
The Pearson’s correlation between fruit biomass and proteins expressed and final concentrations of Ni in soil and in tomatoes (Table 7) highlighted significant correlation between Ni in tomato and thaumatin (r=0.40, P<0.01) and Ni in soil and thaumatin (r=0.51, P<0.01).