Article pubs.acs.org/JAFC
Effectiveness of Iron Ethylenediamine‑N,N′‑bis(hydroxyphenylacetic) Acid (o,o‑EDDHA/Fe3+) Formulations with Different Ratios of Meso and d,l‑Racemic Isomers as Iron Fertilizers Sara Alcañiz, Juana D. Jordá, and Mar Cerdán* Agrochemistry and Biochemistry Department, Faculty of Sciences, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain ABSTRACT: Two o,o-EDDHA/Fe3+ formulations (meso, 93.5% w/w of meso isomer; and d,l-racemic, 91.3% w/w of d,lracemic mixture) were prepared, and their efficacy to avoid or to relieve iron deficiency in Fe-sufficient and Fe-deficient tomato plants grown on hydroponic solution was compared with that of the current o,o-EDDHA/Fe3+ formulations (50% of meso and d,l-racemic isomers). The effectiveness of the three o,o-EDDHA/Fe3+ formulations was different depending on the iron nutritional status of plants. The three o,o-EDDHA/Fe3+ formulations tested were effective in preventing iron chlorosis in healthy plants. However, the higher the meso concentration in the formulations, the higher the effectiveness in the recovery of iron chlorotic plants from iron deficiency. Accordingly, o,o-EDDHA/Fe3+ formulations rich in meso isomer are recommended in hydroponic systems. KEYWORDS: iron chelates, Lycopersicon esculentum Mill., o,o-EDDHA/Fe3+ isomers, iron uptake, iron nutrition, hydroponic culture
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INTRODUCTION
isomers in supplying Fe to plants grown in both hydroponic and soil conditions. Ryskievich and Boka14 studied the effectiveness of separated d,l-racemic and meso isomers in soil application. They found that both were effective in providing bean plants with Fe. Schenkelveld et al.15,16 also observed that d,l-racemic and meso o,o-EDDHA/Fe3+ isomers were effective in supplying soil-grown plants with Fe, approximately to the same extent. These authors concluded that there was no incentive for altering the ratio between d,lracemic and meso o,o-EDDHA/Fe3+ isomers in current commercial o,o-EDDHA/Fe3+ formulations for soil application. The ability of o,o-EDDHA/Fe3+ isomers to supply Fe to plants in hydroponic systems depends widely on the pH of the solution and the occurrence of ions that compete with iron for the chelating agent or with the chelating agent for the iron.17,18 Both d,l-racemic and meso o,o-EDDHA/Fe3+ isomers are completely decomposed at pH 1.0, and at pH values 90% of both isomers at pH 6 in the absence of ions competitors, but slower if Cu is present.18,20 Cerdán et al.21 suggested that the effectiveness of o,o-EDDHA/Fe3+ as iron fertilizer in hydroponic systems may be increased by selecting one of the isomers or by changing the proportion of each one in the current mixes (1:1 ratio). In agreement with that, these authors observed a higher uptake of iron from meso isomer than d,l-racemic mixture by strategy I plants when they grew in nutrient solution that contained 50% of each o,o-EDDHA/Fe3+ isomers.21 Moreover, Á lvarez-
Although iron (Fe) is usually the most abundant plant nutrient in the mineral phase of soils,1 Fe deficiency is a common nutritional problem in plants growing in calcareous and/or alkaline soils due to the low solubility of Fe under the soil condition prevailing in these environments.2 Likewise, alkalinity in irrigation waters, which is associated with the presence of carbonate species, reduces Fe availability in the growing media.3 Dicotyledonous species (strategy I plants) respond to Fe deficiency through the release of root exudates as protons (via an increase of activity of plasma membrane H+-ATPase),4 organic acids, and phenolic compounds,5 followed by the reduction of Fe3+ to Fe2+ by a Fe3+-chelate reductase (FRO)6 and the formation of subapical swollen root tips with abundant root.7,8 However, high bicarbonate concentration in calcareous soils and irrigation waters inhibits the Fe uptake mechanism in strategy I plants and Fe translocation into shoots and leaves,9,10 causing severe iron deficiency symptoms in plants, which require an external application of iron. Nowadays, the application of synthetic Fe chelates is the most common agricultural practice for avoiding and relieving iron deficiency. Iron ethylenediamine-N,N′-bis(hydroxyphenylacetic) acid (o,o-EDDHA/Fe3+) is the most effective synthetic Fe chelate under neutral and alkaline conditions.11,12 The chelating agent o,o-EDDHA is constituted of two diastereomers, a meso form and a racemic mixture, that when linked to iron yield two groups of isomers also known as d,l-racemic and meso isomers in a 1:1 ratio. These isomers show different physical−chemical properties,13 including different stability constants of the complexes with Fe (log K values of 35.86 and 34.15 for the d,l-racemic and meso forms, respectively13) and, as a consequence, so does their ability to preserve Fe in solution and deliver it to the plant. During recent years, considerable effort has been made to establish the efficacy of the individual o,o-EDDHA/Fe3+ © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
July 25, 2016 December 15, 2016 December 19, 2016 December 19, 2016 DOI: 10.1021/acs.jafc.6b03274 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Fernández et al.22 showed that only the meso isomer was depleted from the nutrient solution containing both isomers (in a 1:1 ratio) when o,o-EDDHA/Fe3+ was used as iron source for sunflower plants. Likewise, Lucena and Chaney23,24 found both reduction and Fe concentration in the xylem sap of cucumber plants were higher for the less stable o,o-EDDHA/Fe3+ isomer, although reductase activity was similar for both isomers at pH 6 and lower for the meso isomer at pH 7.5. Orera et al.25 studied the uptake, movement, and distribution of Fe from d,l-racemic and meso o,o-EDDHA/Fe3+ when applied simultaneously to the roots of Fe-deficient strategy I plants. These authors observed that the meso form was the major contributor to deliver Fe to plants in nutrient solution, with rates of ferric chelate reductase, xylem transport, and uptake 2-fold higher than those found for d,l-racemic isomer. The aim of this research was to test the effectiveness of the three o,o-EDDHA/ Fe3+ formulations, FeM (93.5% purity on meso), FeQ (47.3% d,l-racemic and 52.7% meso), and FeR (91.3% purity on d,lracemic), in avoiding iron chlorosis in healthy tomato plants and relieving iron deficiency in iron chlorotic plants.
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After the cotyledons emerged, the seedlings were allowed to grow in the same chamber with a day/night regimen of 16/8 h, a temperature of 27/18 °C with a 16 h photoperiod at a light intensity of 250 μE· m−2·s−1 of photosynthetically active radiation at the level of the lowest leaf, and 70% relative humidity, and they were irrigated with a halfstrength nutrient solution.29 In this nutrient solution, Fe was added as 3.58 × 10−5 M Fe-EDTA for Fe-sufficient plants, and no iron was added to the nutrient solution for Fe-deficient plants. When the seedlings reached a height of 15 cm, they were transferred to 100 mL black plastic pots (one plant/pot) containing 90 mL of continuously aerated nutrient solution with the following composition:29 3.5 × 10−3 M Ca(NO3)2·4H2O, 1.25 × 10−3 M MgSO4·7H2O, 4.5 × 10−3 M KNO3, 7.5 × 10−4 M K2SO4, 1.5 × 10−3 M KH2PO4, 5.0 × 10−4 M NH4NO3, 3.14 × 10−7 M CuSO4·5H2O, 1.36 × 10−6 M ZnSO4·7H2O, 1.27 × 10−5 M MnSO4·H2O, 5.95 × 10−8 M (NH4)6Mo7O24·4H2O, and 4.63 × 10−5 M H3BO4. The pH of the nutrient solution was adjusted to 6.5 with KOH (Panreac, analytical grade). For both Fe-sufficient plants and Fe-deficient plants, iron (35.8 μM) was added to the nutrient solution as one of the three o,oEDDHA/Fe3+ formulations synthesized (FeM, FeQ, or FeR). The pots were placed in a growth chamber with the same environmental conditions established during seedling development. The plants were grown for 15 days. Every day, the losses of volume in the nutrient solutions were replaced with deionized water and, then, plant weight, pH, and EC were measured. Nutrient solution was renewed when the EC value was P.
EDDHA/Fe3+ formulations suggests that Fe uptake by Fesufficient plants is related to the meso concentration in nutrient solution. Because overall Fe uptake by Fe-sufficient plants in FeM treatment was higher and/or faster than in FeR and FeQ treatments, Fe uptake was not yet maximal in FeR and FeQ. Therefore, it can be concluded that the FeM formulation was more effective in facilitating Fe to Fe-sufficient plants because of the higher meso isomer concentration in this formulation. According to the stabilities predicted by Yunta et al.13 from thermodynamic data, the lower stability of the meso isomer (pK = 34.15) with regard to d,l-racemic isomer (pK = 35.86) should facilitate Fe3+ reduction by FCR enzyme and the breakdown of the iron chelate at the root surface.23,24 Moreover, the recent findings of Orera et al.25 and Tomasi et al.38 show that the higher acquisition of Fe from meso isomer could also be related to a more efficient translocation and allocation in the leaf tissue of iron provided by this isomer. In this respect, Zamboni et al.39 observed that the concentration of Fe taken up by Fe-sufficient tomato plants was strongly influenced by the nature of the chelating agent. These authors found the tomato plant supplied with Fe complexed to humic substances reached values about 2 times higher than those measured in response to the supply with Fe-citrate and Fephytosiderophores.39 With regard to the behavior of meso and d,l-racemic isomers, Table 1 shows that the drop of d,l-racemic isomer concentration in FeQ, FeM, and FeR nutrient solutions at 7 days was very low, practically negligible. These results indicated that Fe uptake by Fe-sufficient tomato plants at 7 days of plant growth came mainly from the meso isomer, regardless of the o,o-EDDHA/Fe3+ formulation used (Table 1). These results are in agreement with the findings of Cerdán et al.21 for tomato plants and those of Alvaréz-Fernández et al.22 for sunflower, which showed that only meso isomer is depleted from a nutrient solution with 50% of both meso and d,l-racemic isomers when o,o-EDDHA/Fe3+ was used as Fe source for plants. For FeM solution, the depletion of d,l-racemic in the nutrient solution remained very low for all sampling times (0.06 ± 0.01 μmol at 7 days, 0.11 ± 0.02 μmol at 14 days; Table 1), although there was a tendency to increase slightly with time. However, the depletion of d,l-racemic isomer in FeQ nutrient
Organic Anions. Organic anions were measured according to the method of López-Millán et al.35 Two hundred milligrams of frozen root material was crushed with 8 mM H2SO4 and heated in a bath at 100 °C for 10 min. Then, the samples were filtered through a 0.2 μm polyvinyl filter, made up to volume (2 mL) with 8 mM H2SO4, and analyzed by HPLC. Statistical Analysis. Data were statistically evaluated by analysis of variance (ANOVA) with the SPSS 15.0 program. Means were compared using Duncan’s test at α < 0.05.
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RESULTS AND DISCUSSION
In this study, soluble Fe and chelated Fe concentrations in samples of nutrient solutions at 7 and 14 days of plant growth were measured. No statistically differences between soluble Fe (measured by ICP-OES) and o,o-EDDHA/Fe3+ (measured by HPLC), were found (data not shown); consequently, all Fe remaining in nutrient solution was chelated Fe by o,o-EDDHA. Numerous studies have demonstrated that Fe is not displaced from o,o-EDDHA by other ions in nutrient solutions unless sharp changes in pH occur.17−20,36 Moreover o,o-EDDHA/Fe3+ is biologically reduced but unreactive in photochemically or abiotic redox processes;37 that is, the depletion of o,o-EDDHA/ Fe3+ in the nutrient solution should be owed to plant occurrence. This was also confirmed in the no-plant control used in the experiments, in which no changes in o,o-EDDHA/ Fe3+ were observed. Accordingly, the iron uptake from the three chelate formulations tested (FeM, FeQ, and FeR) by Fesufficient and Fe-deficient tomato plants was calculated as the depletion of o,o-EDDHA/Fe3+ and meso and d,l-racemic o,oEDDHA/Fe3+ isomers from the nutrient solution at 7 and 14 days after transplantation (Table 1). Effectiveness of o,o-EDDHA/Fe3+ Formulations with Different Ratios of Meso and d,l-Racemic Isomers To Avoid Iron Deficiency in Healthy Tomato Plants. For Fesufficient plants, there were statistically significant differences in the drop of o,o-EDDHA/Fe3+ concentration depending on the o,o-EDDHA/Fe3+ formulation used (Table 1). The depletion of o,o-EDDHA/Fe3+ in the FeM nutrient solution was statistically higher than those found in FeQ and FeR nutrient solutions for each sampling time (Table 1). The increase in the depletion of o,o-EDDHA/Fe3+ with increasing meso isomer content in o,oC
DOI: 10.1021/acs.jafc.6b03274 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. pH variation in FeM (93.5% purity on meso), FeQ (47.3% d,l-racemic and 52.7% meso), and FeR (91.3% purity on d,l-racemic) nutrient solutions and no-plant control (Ctrl) for (A) Fe-sufficient plants and (B) Fe-deficient plants. Values are means ± SE.
Figure 2. Fresh weight changes in tomato plants grown in FeM (93.5% purity on meso), FeQ (47.3% d,l-racemic and 52.7% meso), or FeR (91.3% purity on d,l-racemic) nutrient solutions: (A) Fe-sufficient plants; (B) Fe-deficient plants.
Table 2. Effect of FeM, FeQ, and FeR Formulations on Root FCR Activity, Total Chlorophyll Concentration, and Electrolyte Leakage of Fe-Sufficient and Fe-Deficient Tomato Plants Grown under Hydroponic Conditions after 15 Days of Plant Growtha EDDHA/Fe3+ formulation
root FCR activity (μmol Fe(II)·g−1FW·h−1)
Fe-sufficient plants
FeM FeQ FeR signifb
0.9 ± 0.2 1.1 ± 0.3 1.1 ± 0.1 ns
Fe-deficient plants
FeM FeQ FeR signif
1.6 ± 0.1 b 10 ± 1 a 16.0 ± 0.6 c ***
experiment
total chlorophyll (mg·g−1 FW) 17.5 ± 0.5 17.2 ± 0.2 17.8 ± 0.6 ns 17.7 ± 0.5 a 15.6 ± 0.6 b 8±1c ***
electrolyte leakage EC1/EC2 (%) 76 ± 3 69 ± 5 67 ± 5 ns 73 ± 5 b 90 ± 2 a 86 ± 2 a ***
a Data are means ± SE. Different letters within the same column denote significant differences between the treatments according to Duncan’s test (P < 5%). bSignificance: ns, P > 5%; *, P < 5%; **, P < 1%; ***, P < 0.1%.
the depletion of d,l-racemic o,o-EDDHA/Fe3+ isomer in FeR nutrient solution. As a result, Fe-sufficient tomato plants took up preferentially Fe from the meso o,o-EDDHA/Fe3+ isomer as compared to the d,l-racemic one, regardless of the meso:d,lracemic ratio in the o,o-EDDHA/Fe3+ formulation tested. The pH variations in the FeM, FeQ, and FeR nutrient solutions for Fe-sufficient plants and no-plant control are presented in Figure 1A. As expected, the pH of the nutrient solution for no-plant control leveled off throughout the complete assay (Figure 1A). The behavior of the pH in the nutrient solutions was similar for the three chelate formulations studied (Figure 1A). The pH value increased gradually during the first 7 days of crop (Figure 1A). Nutrient solution was then renewed, and pH reached a maximum after 3 days (10 cropping days). After that, it dropped to the initial pH value of the
solution was gradually raised throughout the test period (0.08 ± 0.02 μmol at 7 days, 0.39 ± 0.08 μmol at 14 days; Table 1). Despite this, Fe-sufficient plants grown in the FeQ nutrient solution preferentially took iron from meso isomer (0.8 ± 0.1 μmol from meso and 0.39 ± 0.08 μmol from d,l-racemic) (Table 1). For the FeR formulation, all of the meso o,oEDDHA/Fe3+ isomer present as residue in this formulation was taken up. As in the FeQ nutrient solution, the depletion of d,lracemic isomer gradually increased during the sampling time. This behavior suggests that the amount of Fe3+ provided from meso isomer present in the FeR formulation at the beginning of the assay was enough to fully cover the iron requirements of tomato plants and the Fe uptake from the d,l-racemic isomer was practically negligible. However, the increased iron requirements during plant growth resulted in the gradual increase of D
DOI: 10.1021/acs.jafc.6b03274 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 3. Effect of FeM, FeQ, and FeR Formulations on Organic Anions Concentration in Roots of Fe-Sufficient and FeDeficient Tomato Plants Grown under Hydroponic Conditions after 15 Days of Plant Growtha experiment
EDDHA/Fe3+ formulation
citrate (μmol·g−1 FW)
malate (μmol·g−1 FW)
succinate (μmol·g−1 FW)
fumarate (μmol·g−1 FW)
Fe-sufficient plants
FeM FeQ FeR signifb
23 ± 9 20 ± 7 13 ± 3 ns
10 ± 4 10 ± 2 7±1 ns
15 ± 8 15 ± 3 8.7 ± 0.6 ns
1.7 ± 0.4 2.2 ± 0.8 1.1 ± 0.2 ns
Fe-deficient plants
FeM FeQ FeR signif
10 ± 5 8±2 7±3 ns
5±2 4±1 7±3 ns
4±2b 4.0 ± 0.9 b 8.4 ± 0.8 a ***
0.6 ± 0.4 b 0.6 ± 0.3 b 1.2 ± 0.3 a ***
a Data are means ± SE. Different letters within the same column denote significant differences between the treatments according to the Duncan test (P < 5%); bSignificance: ns, P > 5%; *, P < 5%; **, P < 1%; ***, P < 0.1%.
study of Schenkelveld et al.15,16 when they tested the effectiveness of separated d,l-racemic and meso isomers providing Fe to Fe-deficient plants in soil application. According to the data shown in Table 1, the depletion of o,oEDDHA/Fe3+ in FeQ and FeR nutrient solutions of Fedeficient plants was higher than that found for Fe-sufficient plants grown with the same treatments (Table 1). This behavior was mainly observed at the first sampling time (7 days), whereas these differences were much smaller at 14 days (Table 1). The highest drop of o,o-EDDHA/Fe3+ concentration in FeQ and FeR nutrient solutions for Fe-deficient plants regarding Fe-sufficient plants could be correlated with the decrease of pH observed for Fe-deficient plants during the first days of the assay (Figure 1B). According to Gómez-Gallego et al.,37 at the more acid pH in the vicinity of the roots, the o,oEDDHA/Fe3+ chelate could lead to an open hexacoordinate species (FeHL), which is a better substrate for FCR enzyme and, hence, the enzymatic reduction of Fe3+ to Fe2+ would be more effective. Table 2 shows that FCR activity of Fe-deficient plants grown in the FeQ and FeR solutions at 14 days was 10and 16-fold higher than that observed in Fe-sufficient plant developed in the same solution. This is indicative that the activation of mechanisms of response of strategy I plant was turned on.6 Succinate and fumarate also increased in plants grown in FeR solutions (Table 3). Organic acids are known to increase as a consequence of Fe deficiency.5 In contrast, the depletion of o,o-EDDHA/Fe3+ in FeM nutrient solutions at 7 and 14 days after transplantation was similar for both Fesufficient and Fe-deficient tomato plants (Table 1). Moreover, the root FCR activity of Fe-deficient plants grown in FeM nutrient solution was only 1.8-fold higher than that observed for Fe-sufficient plants treated with the same formulation (Table 2). This behavior indicates that the responses to Fe shortage could be switched off in Fe-deficient plants developed in FeM solution. Therefore, these tomato plants could have recovered from iron deficiency more quickly that those treated with the FeQ and FeR formulations, probably due to the higher capability of Fe-deficient plants to utilize iron from meso isomer.23−25,38 This hypothesis was supported by the values of leaf chlorophyll content, the root membrane permeability (Table 2), and the fresh weight increase (Figure 2B), which were statistically significantly higher for the Fe-deficient plants treated with FeM formulation than for Fe-deficient plants treated with FeQ and FeR. Moreover, when the chlorophyll content of the Fe-sufficient plants and Fe-deficient plants develop in FeM were compared, it was observed that the order of magnitude of this parameter was similar for both Fe-deficient
nutrient solution (Figure 1A). The pH of the three nutrient solutions decreased at the end of the assay, although the pH values for Fe-sufficient plants and no-plant control were statistically similar (Figure 1A). This pH changes may be explained by nutrient uptake, alterations in the cation−anion balance, and CO2 accumulation from respiration processes, which may be especially relevant at the end of the experiment owing to plant weight.40 Consequently, the acidification of the rhizosphere by the activation of the H+-ATPase as a strategy I response against an iron deficiency situation4 was not observed in any nutrient solution. Figure 2A shows the behavior of the fresh weight of plants developed in FeM, FeQ, and FeR nutrient solutions throughout the assay. The increase of plant weight during the experiment was statistically similar for the three tested formulations (Figure 2A). All formulations resulted in well-developed plants. On the other hand, significant differences in root FCR capacity, leaf chlorophyll content electrolyte leakage (Table 2), and root organic anion concentration (Table 3) between the three o,o-EDDHA/Fe3+ formulations tested (FeM, FeQ, and FeR) were not found. Moreover, these plants did not show any visual symptom of iron deficiency. This suggests that the Fesufficient plants developed in the FeQ and FeR nutrient solutions were not iron-stressed plants, although the depletion of o,o-EDDHA/Fe3+ in these nutrient solutions was approximately half of that found in the FeM solutions (Table 1). The higher decrease in FeM concentration must be facilitated by a lower stability constant of the meso isomer.13 As no Fe precipitation or turbidity was observed in FeM nutrient solutions, the excess of Fe from FeM might be stored in tomato plants. Additional experiments would be necessary to test the hypothesis. Effectiveness of o,o-EDDHA/Fe3+ Formulations with Different Ratios of Meso and d,l-Racemic Isomers To Relieve Iron Deficiency in Iron Chlorotic Plants. The data of the depletion of o,o-EDDHA/Fe3+ in the FeM, FeQ, and FeR nutrient solutions at 7 and 14 days of Fe-deficient plant growth are shown in Table 1. As can be seen in Table 1, the behavior of Fe-deficient plants was different from that observed for Fe-sufficient plants. For no sampling times were significant differences in the drop of o,o-EDDHA/Fe3+ concentration in the FeM, FeR, and FeQ nutrient solutions observed (Table 1). These results indicate that both meso and d,l-racemic isomers contributed to Fe uptake by Fe-deficient plants, as shown from the fact that in the three formulations tested (FeQ, FeM, and FeR), the depletion of o,o-EDDHA/Fe3+ was statistically similar (Table 1). This is in agreement with the conclusion from the E
DOI: 10.1021/acs.jafc.6b03274 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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it may have influenced plant growth. The evidence suggests that the effectiveness of commercial o,o-EDDHA/Fe3+ formulations in hydroponic system could be increased, changing the ratio between d,l-racemic and meso o,o-EDDHA/Fe3+ isomers depending on the pre-existing iron nutritional status of the crops.
and Fe-sufficient plants (Table 2). With reference to FeQ and FeR nutrient solutions, it should be noted that chlorophyll content in the leaves of Fe-deficient plants developed in FeQ solution was 2 times higher than those grown in FeR solution (Table 2). As the depletion of o,o-EDDHA/Fe3+ was similar in the three formulations tested (Table 1), these results suggest that the iron from meso isomer was better assimilated by Fedeficient plants25,38 and that Fe from d,l-racemic isomer might be considered as low-available Fe, owing to its high stability constant.13 Although the decrease of Fe concentration in FeM and FeR solutions was similar and no Fe-chlorosis symptoms were observed at the end of the experiment in any case, Fedeficient plants in FeR solutions had more active strategy I uptake mechanisms, and this might affect the plant growth (Figure 2B) With respect to the depletion of meso or d,l-racemic o,oEDDHA/Fe3+ isomers in the nutrient solutions, Fe-deficient plants showed a behavior similar to those found for Fesufficient plants (Table 1). Consequently, as for Fe-sufficient plants, Fe-deficient plants preferentially took iron from the meso isomer (Table 1). When plants are subjected to environmental stresses such as salinity, drought, temperature extremes, or mineral deficiency, the balance between the production of reactive oxygen species (ROS) and the quenching activity of antioxidants is upset, often resulting in oxidative damage.41 Iron deficiency is reported to induce oxidative stress in plants, because of ROS production.42 The induced generation of O2− and H2O2 leads to oxidative stress because of an incomplete reduction of oxygen,43 which results in membrane lipid peroxidation, causing membrane damage and leakage of electrolytes. 44 The membrane permeability of the Fe-deficient plants grown in FeM solution was statistically significantly lower than that of the Fe-deficient plants developed in the FeQ and FeR solutions (Table 2). Then, these results could imply a lower damage to the cell membrane integrity of Fe-deficient plants grown in FeM solutions with regard to those developed in FeQ and FeR solutions. By comparing the data about the membrane permeability of the Fe-sufficient and Fe-deficient plants grown in the FeM formulation, it was found that both parameters were similar (Table 2). This supports the hypothesis that Fe-deficient plants developed in FeM formulations could be subject to lower oxidative damage, owing to a better recovery of the iron nutritional status of Fedeficient plants grown in FeM formulations with regard to those developed in FeQ and FeR solutions. On the basis of the results obtained, it was concluded that the effectiveness of the three o,o-EDDHA/Fe3+ formulations tested as iron fertilizer depended on the iron nutritional status of strategy I plants. The three o,o-EDDHA/Fe3+ formulations studied avoided iron deficiency in healthy plants. A longer term study is required to establish if the higher drop of o,o-EDDHA/ Fe3+ concentration in FeM nutrient solution of Fe-sufficient tomato plants grown in FeM solutions could produce a higher Fe uptake and consequently even an iron toxicity or whether, on the contrary, the lower depletion of o,o-EDDHA/Fe3+ in FeQ and FeR nutrient solutions of Fe-sufficient tomato plants could cause an iron deficiency situation. The effectiveness of o,o-EDDHA/Fe3+ formulations to recover iron chlorotic plants from iron deficiency was increased with the quantity of meso isomer in the o,o-EDDHA/Fe3+ formulation. The intensity in the activation of the strategy I mechanisms in Fe-deficient plants was related to the stability of the Fe chelate supplied, and
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AUTHOR INFORMATION
Corresponding Author
*(M.C.) Phone: +34 965 903400, ext. 3116. Fax: +34 965 909955. E-mail:
[email protected]. ORCID
Mar Cerdán: 0000-0002-0636-1144 Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED o,o-EDDHA, ethylenediamine-N,N′-bis(o-hydroxyhenylacetic)acid; o,o-EDDHA/Fe3+, iron ethylenediamine-N,N′-bis(o-hydroxyphenylacetic) acid; FeM, o,o-EDDHA/Fe3+ solution with a 93.5% purity of meso isomer; FeR, o,o-EDDHA/Fe3+ with a 91.3% purity of d,l-racemic mixture; FeQ, o,o-EDDHA/Fe3+ solution containing 47.3% of d,l-racemic mixture and 52.7% of meso isomer; HPLC, high-performance liquid chromatography; ICP-OES, inductively coupled plasma-optical emission spectroscopy
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REFERENCES
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DOI: 10.1021/acs.jafc.6b03274 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jafc.6b03274 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
