Composition of the Fe sub-products used
The Feoxi sub-product (70.0% Fe) was found to contain (using EDX) 82.2% FeO (wüstite), 2.3% Fe2O3 (hematite) and 7.9% Fe3O4 (magnetite) (Figure S2). The Feore sub-product (60.5% Fe) was found to contain 36.5% FeS2 (pyrite), 33.2% hematite, 14.1% magnetite, 4.9% wüstite and 4.9% Fe0.4Mg0.6O (magnesiowüstite) (Figure S3).
Changes in leaf SPAD with Fe and thiol treatments
A picture of the plants at day 50 is shown for some treatments in Figure S4. The chlorosis level of the plants growing with no Fe in the absence of thiols was 2 (some chlorosis in the canopy, as in Merry et al. 2022). In the absence of thiols (grey bars in Fig. 1) all three Fe-containing products led to increases in the SPAD values of young leaves when compared to the zero Fe treatment (in the range from 1.1- to 1.4-fold). In plants grown with no Fe added, the SPAD index increased when GSH, DTT and PhSH were applied (in the range from 1.1- to 1.3-fold). In the case of plants treated with Fe-EDDHA, the SPAD also increased with all thiol compounds tested (in the range from 1.1- to 1.2-fold). However, in plants treated with Feoxi and Feore, the SPAD did not increase significantly with the thiol treatments. When GSH was applied, all three Fe sources led to increases in the SPAD values when compared to the zero Fe treatment. However, when DTT was applied, Fe treatments did not lead to increases in SPAD, and when PhSH was applied, the application of Feoxi and Feore, but not that of Fe(III)-EDDHA, led to increases in SPAD.
Changes in biomass and plant height with Fe and thiol treatments
Changes observed in the shoot and root FW with Fe and thiol treatments are shown in Figure S5 and described in detail in the Supplementary materials file.
When no thiols were added, the only shoot DW change found was a decrease for Feoxi (7%; grey bars in Fig. 2A). In plants grown with zero Fe, the DW was not changed when any of the thiols were applied. In plants treated with the Fe-chelate, the DW increased when GSH and PhSH were applied (1.2-fold). In plants treated with Feoxi and Feore, the DW decreased only with DTT and PhSH (by 20 and 16%, and 28 and 11%, respectively). When GSH, DTT and PhSH were applied with the Fe-chelate, increases in shoot DW were found when compared to the zero Fe treatment. Decreases in the shoot DW were observed when any of the thiols were applied with Feoxi and when DTT and PhSH were applied with Feore.
When no thiols were added, all Fe sources led to increases in root DW (1.5- to 1.7-fold; grey bars in Fig. 2B). In plants grown with zero Fe, the DW increased when GSH and DTT were applied (1.1- and 1.3-fold, respectively). In plants treated with Fe-EDDHA, the DW decreased with GSH and DTT (by 18 and 12% respectively) and increased with PhSH (1.3-fold). In plants treated with Feoxi, the DW increased with DTT and PhSH (1.1-fold,) and decreased slightly with GSH (by 3%). In plants treated with Feore, the root DW decreased with GSH and DTT (by 11 and 14%, respectively) and increased with PhSH (1.1-fold). For any thiol treatment, addition of all Fe sources led to increases in root DW.
Regarding plant height, when no thiols were added all Fe sources led to increases in this parameter (1.9- to 2.1-fold; grey bars in Fig. 2C). In plants grown with zero Fe, plant height increased only when GSH and PhSH were applied (1.4- and 1.2-fold, respectively). In plants treated with any of the Fe products, plant height decreased when thiols were applied (by 15–21% for the Fe-chelate, 37–41% for Feoxi and 28–42% for Feore). For any thiol treatment, supplementation with the Fe-chelate led to increases in plant height. Other significant changes in plant height were decreases when GSH was supplemented with Feore and increases when DTT was supplemented with Feoxi and Feore.
Changes in Fe concentrations and contents in leaves, stems and roots with Fe and thiol treatments
In the absence of thiols, using the Fe-chelate and Feoxi led to increases in the Fe concentrations in young leaves (1.3- and 1.2-fold, respectively; grey bars in Fig. 3A) In young leaves of plants grown with zero Fe, Fe concentrations decreased when thiols were applied (by 13–35%). In plants treated with the three Fe sources, Fe concentrations increased with DTT (in the range from 1.1- to 1.7-fold), and decreased in the cases of the Fe-chelate and Feore with the other two thiols (by 18–38% and 21–26% respectively), and in the case of Feoxi with PhSH (by 34%). With any thiol treatment, application of the three Fe sources led to increases in the Fe concentration when compared to the zero Fe treatment, with the only exception of the treatment with GSH with Feore, where there was no effect.
When no thiols were applied, all Fe compounds led to increases in the Fe concentration in developed leaves when compared to the zero Fe control (1.4- to 1.9-fold; grey bars in Fig. 3B). In developed leaves of plants grown with zero Fe, Fe concentrations increased when thiols were applied (in the range from 1.1- to 1.5-fold). In the case of plants treated with Fe(III)-EDDHA, the Fe concentration increased with DTT (1.2-fold), and decreased with PhSH (by 11%). In plants treated with Feoxi, the leaf Fe concentration increased when GSH and PhSH were applied (2.0- and 1.5-fold, respectively). In plants treated with Feore, the Fe concentration increased with all thiols tested, with PhSH having the largest effect (1.1-, 1.2- and 1.7-fold). For any thiol treatment, all Fe compounds led to increases in the leaf Fe concentration when compared to the zero Fe control.
Regarding the Fe concentrations in stems, when no thiols were added all Fe compounds led to increases in this parameter (1.3- to 1.6-fold; grey bars in Fig. 3C) In stems of plants grown with zero Fe, the Fe concentrations decreased slightly when DTT was applied (by 4%) and increased with PhSH (1.1-fold). In plants treated with Fe(III)-EDDHA, the Fe concentration increased with DTT (1.1-fold), and decreased with GSH and PhSH (by 4% and 21%, respectively). In plants treated with Feoxi, the leaf Fe concentration increased with all thiols (1.2-fold), whereas in plants treated with Feore, the Fe concentration increased with DTT and PhSH (1.3-fold). For any thiol treatment, all Fe compounds led to increases in the leaf Fe concentration when compared to the zero Fe control.
In the absence of thiols, only the Fe-chelate led to increases in the Fe concentration in roots when compared to the zero Fe control (2.4-fold; grey bars in Fig. 3D). In plants grown with zero Fe, the Fe concentration only increased with GSH and PhSH (1.3- and 1.6-fold, respectively). In the case of plants treated with Fe(III)-EDDHA, the Fe concentration decreased with all thiols (by 10–27%). Conversely, in plants treated with Feoxi and Feore, the Fe concentration increased markedly with all thiols (from 1.5- to 1.8-fold and 1.4- to 1.7-fold, respectively). When any of the thiols were applied, all Fe treatments led to increases in the root Fe concentration when compared to the zero Fe control.
When no thiols were applied, the only increase in leaf extractable Fe was when using Feoxi. (1.2-fold; grey bars in Figure S6). In plants grown with zero Fe, the extractable Fe (with 1,10-o-phenanthroline) increased only when GSH was applied (1.3-fold). In the case of plants treated with Fe(III)-EDDHA, the extractable Fe increased only with DTT and PhSH (1.4- and 1.1-fold, respectively). In plants treated with Feoxi, the extractable Fe decreased only with PhSH (23%), and in those treated with Feore, the extractable Fe increased with GSH and PhSH (1.6- and 1.4-fold, respectively), but decreased with DTT (by 13%). When GSH was applied, decreases and increases in leaf extractable Fe were found with the Fe-chelate and Feore, respectively. When DTT was used, increases in leaf extractable Fe were found with the Fe-chelate and Feox, and decreases with Feore. Finally, when PhSH was used, increases in leaf extractable Fe were found with Feore, and decreases with Feoxi.
Changes in iron contents with Fe and thiol treatments
When no thiols were added, all Fe compounds led to increases in the Fe contents in young leaves when compared to the zero Fe control (1.1- to 1.3-fold; grey bars in Fig. 4A). In leaves of plants grown with zero Fe, the Fe contents decreased when all thiols were applied (by 13–35%). In plants treated with Fe(III)-EDDHA, the leaf Fe content increased with DTT (1.1-fold), and decreased with GSH and PhSH (by 18 and 38%, respectively). In plants treated with Feoxi, the leaf Fe content increased when DTT was applied (1.1-fold) and decreased with PhSH (by 34%). In plants treated with Feore, the leaf Fe content decreased with GSH and PhSH (by 21–26%) and increased with DTT (1.7-fold). For any thiol treatment, all Fe treatments led to increases in the young leaf Fe content when compared to the zero Fe control, with the only exception of Feore applied in combination with GSH.
In the absence of thiols, all Fe compounds led to increases in the Fe contents in developed leaves when compared to the zero Fe control (1.4- to 1.9-fold; grey bars in Fig. 4B). In plants grown with zero Fe, the leaf Fe contents increased when thiols were applied (from 1.1- to 1.5-fold). In the case of plants treated with the Fe-chelate, the leaf Fe content showed increases and decreases with DTT and PhSH (1.1-fold and 11%, respectively). In plants treated with Feoxi, the leaf Fe contents increased with GSH and PhSH (2.0- and 1.5-fold, respectively). In plants treated with the Feore, the leaf Fe contents increased with all thiols applied (from 1.1- to 1.7-fold). With any thiol treatments, increases in developed leaf Fe contents were found when using all three Fe compounds.
Regarding the Fe contents in stems, when no thiols were added all Fe compounds led to increases in this parameter when compared to the zero Fe control (1.5- to 2.5-fold; grey bars in Fig. 4C). In stems of plants grown with zero Fe, the Fe contents did not change when thiols were applied. In the case of plants treated with Fe(III)-EDDHA and Feoxi, the Fe content decreased with DTT and PhSH (from 8–22%). In plants treated with Feore, the Fe content only increased with PhSH (1.2-fold). For any thiol treatment, all Fe compounds led to increases in stem Fe contents when compared to the zero Fe control.
When no thiols were added, all Fe compounds led to increases in the Fe contents in roots when compared to the zero Fe control (1.7- to 3.8-fold; grey bars in Fig. 4D). In roots of plants grown with zero Fe, Feoxi and Feore, the Fe content increased with all thiol compounds (from 1.2- to 2.0-fold). In the case of plants treated with the Fe-chelate, the root Fe content decreased with all thiols (by 7–33%). With any thiol treatments, all Fe treatments led to increases in the root Fe concentrations when compared to the zero Fe control.
Changes in S concentrations and contents with Fe and thiol treatments
In the absence of thiols, the Fe-chelate led to decreases in the S concentrations in the young leaves, whereas Feoxi and Feore led to increases in this parameter (16% and from 1.2- to 1.3-fold, respectively; grey bars in Figure S7A). In plants grown with zero Fe, the leaf S concentrations only decreased very slightly with DTT. In plants treated with the Fe-chelate, the S concentrations increased with all thiols (from 1.2- to 1.6-fold). In plants treated with Feoxi, the S concentrations increased 1.4–fold with GSH and 1.1-fold with DTT. In plants treated with Feore, the S concentrations increased with GSH and DTT (from 1.1- to 1.2-fold), and decreased somewhat with PhSH (by 9%). When GSH and DTT were applied, all Fe compounds led to increases in S concentrations, and when PhSH was applied, only Feoxi and Feore led to increases in S concentrations.
Regarding the root S concentrations, when no thiols were applied the Fe-chelate and Feore led to S concentration decreases and increases, respectively (32% and 1.1-fold, respectively; grey bars in Figure S7C). In roots of plants grown with zero Fe, the S concentrations increased when all thiols were applied (ca. 1.2-fold). In plants treated with the Fe-chelate and Feoxi, the root S concentration also increased with all thiols were applied (from 1.4- to 1.8-fold and from 1.1- to 1.4-fold, respectively). In plants treated with Feore, the root S concentration increased 1.5-fold with GSH and 1.1-fold with DTT, and decreased with PhSH (by 6%). When GSH was applied, all three Fe compounds led to increases in root S concentration. When DTT was applied, only the Fe-chelate led to increases in root S concentration, and when PhSH was applied, all Fe compounds led to decreases in S concentrations.
When no thiols were applied, the Fe-chelate and Feore led to young leaf S content decreases and increases, respectively (21% and 1.2-fold, respectively; grey bars in Figure S7B). In leaves of plants grown with zero Fe, the S contents did not change when thiols were applied. In plants treated with the Fe-chelate, the leaf S content increased with all thiols were applied (in the range 1.6- to 2.1-fold). In plants treated with Feoxi, the leaf S contents only increased with GSH (1.4–fold). In plants treated with Feore, the leaf S contents decreased with DTT and PhSH (by 29 and 19%, respectively). When GSH was applied, all three Fe compounds led to increases in S content. When DTT was applied, only the Fe-chelate led to increases in leaf S content, and when PhSH was applied, the Fe-chelate and Feoxi led to increases and decreases in leaf S content, respectively.
In the absence of thiols, Feoxi and Feore led to root S content increases (1.5- and 1.9-fold, respectively; grey bars in Figure S7D). In roots of plants grown with zero Fe, the S contents increased when all thiols were applied (from 1.2- to 1.6-fold). In plants treated with the Fe-chelate and Feoxi, the S contents also increased with all thiols (from 1.3- to 1.8-fold), whereas in plants treated with Feore, the S concentration increased with GSH and PhSH (from 1.1- to 1.3-fold) and decreased with DTT (by 8%). When any thiol was applied, all three Fe compounds led to increases in root S contents.
Changes in antioxidant compounds with Fe and thiol treatments
When no thiols were applied, all three Fe sources led to increases in the leaf tGSH concentrations when compared to the zero Fe treatment (2.1- to 2.9-fold; grey bars in Fig. 5A). In plants grown with zero Fe, Feoxi and Feore, the leaf tGSH concentrations increased with all thiols (from 1.7- to 2.3-fold, 1.4- to 1.6-fold and 1.2- to 2.3-fold, respectively). In plants treated with the Fe-chelate, the leaf tGSH decreased with DTT (by 13%). With any thiol treatment, all three Fe products led to increases in the leaf tGSH.
In the absence of thiols, all three Fe sources led to increases in the leaf tGSSG concentrations when compared to the zero Fe treatment (1.7- to 2.4-fold; grey bars in Fig. 5B). In plants grown with zero Fe, Feoxi and Feore, the leaf tGSSG concentrations increased with all thiols (from 1.1- to 1.7-fold, 1.5- to 1.7-fold and 1.3- to 2.2-fold, respectively). In the case of plants treated with the Fe-chelate, the leaf tGSSG decreased with GSH (by 16%). With any thiol treatment, all three Fe products led to increases in the leaf tGSSG when compared to the zero Fe treatment.
Regarding the leaf Asc concentrations, when no thiols were added all three Fe sources led to decreases in this parameter when compared to the zero Fe control (30–35%; grey bars in Fig. 5E). In plants grown with zero Fe, the Asc concentrations decreased with DTT (by 9%), and much more markedly with GSH and PhSH (by 37–43%) (Fig. 5E). In plants treated with the Fe-chelate, the Asc concentrations had decreases (19%) and increases (1.1-fold) with GSH and DTT, respectively. In plants treated with Feoxi, the Asc concentrations also decreased with GSH and PhSH (by 10 and 28%, respectively), and increased with DTT (1.2-fold). In the case of plants treated with Feore, the Asc concentrations also had decreases with GSH (14%) and increases with DTT and PhSH (1.4- and 1.2-fold, respectively). When GSH was added, the Fe-chelate and Feore led to decreases in the leaf Asc concentrations when compared to the zero Fe control. When DTT was added, the Fe-chelate and Feoxi led to decreases in the Asc concentrations, and when PhSH was added, Feoxi and Feore led to decreases and marked increases in the leaf Asc concentrations.
When no thiols were added, all Fe products led to marked decreases in the root tGSH concentrations when compared to the zero Fe treatment (46–61%; grey bars in Fig. 5C). In plants grown with zero Fe and the Fe-chelate, the root tGSH concentrations decreased with all thiols (by 51–64% and 35–43%, respectively). In plants treated with Feoxi, the root tGSH increased only with GSH (1.2-fold). In plants treated with Feore, the root tGSH increased with GSH and PhSH (1.7- and 1.5-fold, respectively). When any of the thiols were applied, the Fe-chelate led to decreases in the root tGSH. Decreases in the root tGSH were also found with GSH and Feoxi, and increases with GSH and PhSH and Feore.
In the absence of thiols, all three Fe products led to marked decreases in the root tGSSG concentration when compared to the zero Fe treatment (47–61%; grey bars in Fig. 5D). In plants grown with zero Fe and the Fe-chelate, the root tGSSG concentrations decreased with all thiols (by 55–68% and 44–47%, respectively). In plants treated with Feoxi, the root tGSSG decreased with GSH (by 12%). In plants treated with Feore, the root tGSSG increased with GSH and PhSH (1.2- and 1.3-fold, respectively) and decreased with DTT (by 16%). When any of the thiols were applied, the Fe-chelate led to decreases in the root tGSSG when compared to the zero Fe control. Other changes in root tGSSG included decreases when GSH was applied with Feoxi and increases when DTT was applied with Feoxi and when PhSH was applied with Feore.
Changes in antioxidant enzyme activities with Fe and thiol treatments
When no thiols were added, all Fe sources led to large increases in the GR activity in leaf extracts when compared to the zero Fe treatment (2.4-fold; grey bars in Fig. 6A). In plants grown with zero Fe, the GR activity increased with the three thiol compounds (from 1.7-fold to 2.4-fold,). In plants treated with Fe-EDDHA, the GR activity was not changed with any of the thiols. In plants treated with Feoxi and Feore, the GR activity increased with GSH ((1.1-fold) and decreased with DTT (18–31%). When GSH was added, Feoxi and Feore led to minor increases in the GR activity, and when DTT was added, the Fe-chelate and Feoxi led to increases in this parameter. Finally, when PhSH was added, Feoxi and Feore led to small increases in the GR activity when compared to the zero Fe treatment.
In the absence of thiols, all Fe products led to increases in the APX activity in leaf extracts when compared to the zero Fe treatment (1.4- to 1.5-fold; grey bars in Fig. 6B). In plants grown with zero Fe, the APX activity increased with GSH and PhSH (1.4- and 1.7-fold, respectively). In plants treated with Fe-EDDHA, the APX activity increased and decreased with GSH and DTT (1.2.fold and 13%, respectively). In plants treated with Feoxi, the APX activity also decreased with DTT and PhSH (by 39 and 20%, respectively). In plants treated with Feore, the APX activity increased with GSH (1.2-fold) and decreased with DTT and PhSH (by 51 and 19%, respectively). In the case of GSH, the Fe-chelate and Feore, but not Feoxi, led to increases in APX activity when compared to the zero Fe control. When DTT was added, the Fe-chelate led to increases in APX activity, whereas Feoxi and Feore led to decreases in this parameter. When PhSH was added, all three Fe-compounds led to decreases in the leaf APX activity when compared to the zero Fe treatment.
Changes in carboxylates with Fe and thiol treatments
In plants grown in the absence of thiols, all Fe products led to marked decreases in the leaf Cit concentration when compared to the zero Fe control (42–72%; grey bars in Fig. 7A). In plants grown with zero Fe, the Cit concentration increased markedly with all thiol compounds (from 2.1- to 2.9-fold). In plants treated with the Fe-chelate, the Cit concentration increased with DTT and PhSH (1.2-fold). In plants treated with Feoxi the leaf Cit concentration increased with the three thiols (from 1.4- to 1.6-fold). In plants treated with Feore, the leaf Cit concentration increased markedly with GSH and PhSH (2.3- and 1.9-fold, respectively). In all the thiol treatments the three Fe sources led to marked decreases in the leaf Cit concentration when compared to the zero Fe treatment.
When no thiols were added, all Fe products led to increases in the leaf Mal concentration (1.1- to 1.3-fold; grey bars in Fig. 7B). In plants grown with zero Fe, the Mal concentration decreased when any of the thiols were added (by 26–48%). In the case of plants treated with the Fe-chelate, the Mal concentration decreased only with DTT (by 9%). In plants treated with Feoxi and Feore, the Mal concentration also decreased with all thiols (11–28%). In all the thiol treatments the three Fe sources led to increases in the leaf Mal concentration when compared to the zero Fe treatment.
Changes in phytohormones with Fe and thiol treatments
When no thiols were added, all three Fe sources led to major increases in the leaf GA concentration when compared to the zero Fe treatment (4.3- to 5.9-fold; grey bars in Fig. 8A). In plants grown with zero Fe, the leaf GA concentrations showed marked increases when GSH and PhSH were applied (2.3- and 1.9-fold, respectively). In plants treated with the Fe-chelate, the leaf GA concentration decreased by half when any thiol was applied (by 47–51%). In the case of plants treated with Feoxi and Feore, the leaf GA concentration also decreased by half with all thiols applied (by 46–52% and 48–60% respectively). When DTT was added, all three Fe products led to major increases in the leaf GA concentration when compared to the zero Fe treatment. When GSH was added, the Fe-chelate and Feore led to increases and decreases in the leaf GA concentration, respectively, and when PhSH was added the Fe-chelate and Feore led to increases in the leaf GA concentration.
Regarding the leaf ABA concentration, in the absence of thiols all three Fe sources led to small decreases in this parameter when compared to the zero Fe controls (16 to 23%; grey bars in Fig. 8B). In plants grown with zero Fe, the leaf ABA concentration decreased by half with GSH and PhSH and less with DTT (by 10%). In plants treated with any Fe product the leaf ABA concentration did not change with thiols, with the exception of the Feore with PhSH, where leaf ABA decreased by 13%. In plants treated with GSH, 2-fold increases in the leaf ABA concentration were found with all Fe compounds. When DTT was added, Feoxi and Feore led to small decreases in the leaf ABA concentration, and when PhSH was added, all three Fe products led to major increases in the leaf ABA concentration when compared to the zero Fe control.
Changes in DTPA-extractable soil Fe with Fe and thiol treatments
In the absence of thiols, only the Feoxi led to increases in DTPA-extractable Fe (1.2-fold; grey lines in Fig. 9). When plants were grown with zero Fe, the DTPA-extractable soil Fe only increased with GSH (1.3-fold). In plants treated with the Fe-chelate, the DTPA-extractable Fe increased with DTT and PhSH (1.4- and 1.1-fold, respectively). In plants treated with Feoxi, the DTPA-extractable Fe increased with GSH and DTT (1.1-fold), and decreased with PhSH (by 23%). In plants treated with Feore, the DTPA-extractable Fe increased with GSH and PhSH (1.6- and 1.6-fold, respectively), and decreased with DTT (by 13%). When GSH was applied, decreases and increases in DTPA-extractable Fe were found with the Fe-chelate and Feore. When DTT was applied, increases in DTPA-extractable Fe were observed with Fe-chelate and Feoxi, and decreases with Feore. When PhSH was applied, decreases in DTPA-extractable Fe were observed with Feoxi, and increases with Feore.
Solubilization of Fe oxides by thiols
Samples were homogenized in 15 mM MES, pH 6.0, supplemented with 300 µM BPDS, in the absence of thiols. After centrifugation to remove the bulk of the products, the total Fe (total ICP-Fe) in the still slightly opalescent solution was 8.9 and 175.6 mg Fe L− 1 with the Feoxi and Feore, respectively. When Fe was measured by ICP without a strong acid digestion (ICP-Fe), the Fe concentration in the solution was two orders of magnitude lower with Feoxi than with Feore (0.1 and 9.3 mg L− 1, respectively; grey bars in Fig. 10A). In the absence of thiols there was a measurable Fe(II) chelation by BPDS in both products, being larger at 120 min than at 60 min (grey bars in Fig. 10B,C).
The addition of thiols led to large increases in the ICP Fe in solution (measured after acidification), with the increases being higher for DTT in the case of Feoxi and for GSH in the case of Feore (Fig. 10A). The addition of thiols also led to large increases in the Fe(II) chelated by BPDS, which were larger when the incubation time increased (Fig. 10B,C, and Fig S8). The Fe(II) values were higher for Feoxi than for Feore for all thiol compounds and at all incubation times. The highest and lowest solubilized Fe(II) values were for GSH and PhSH, respectively, both when using Feoxi and Feore.
In the absence of thiols, the ICP-Fe accounted for approximately 1% and 5% of the total ICP-Fe (for Feoxi and Feore, respectively), whereas in the presence of thiols, the ICP-Fe accounted for ca. 53% and 64% of the total ICP-Fe (for Feoxi and Feore, respectively). The Fe chelated by BPDS constituted a larger fraction of the ICP-Fe in the case of Feoxi (ca. 1%, 16%, 16% and 9% in the cases of the no thiol treatment, GSH, DTT and PhSH, respectively) than in the case of Feore (< 1% in all cases).