Introduction

Iron is an essential micronutrient for plants. However, in alkaline or calcareous soils, the concentration of soluble Fe is usually far below that is needed for normal plant growth; in fact, most of the Fe in soil is present in inorganic forms, predominantly as goethite, hematite, and ferrihydrite, all poorly available for root uptake under aerobic conditions (Lindsay and Schwab 1982). For their survival, plants can rely on a variety of natural organic ligands, which can, with different levels of efficiency, form complexes with Fe3+ favoring the mobilization of Fe from oxides/hydroxides (Cesco et al. 2000; Tomasi et al. 2013) or from Fe-humates (Gerke 1993; Colombo et al. 2012, 2014). Microbial siderophores (Neilands 1981), plant root exudates, including phytosiderophores (Takagi et al. 1984), organic acids (Römheld 1987; Jones et al. 1996), and phenolic compounds (Cesco et al. 2010; 2012), and humic substances (Stevenson 1994; Chen et al. 2004; Colombo et al. 2012) belong to this group of natural organic ligands. It is interesting to note that more than 95 % of the total plant-available Fe in the soil solution could be represented by this organic Fe pool (van Hees and Lundstrom 2000).

Concerning these natural Fe sources, it has been clearly demonstrated that plants can use them with mechanisms differing for the ligand and/or the plant species: in particular, dicots perform the reduction-based chelate splitting followed by Fe2+ intake (Römheld and Marschner 1986; Pinton et al. 1999a), although direct uptake of the entire Fe complex has been reported in some cases (Wang et al. 1993; Vansuyt et al. 2007; Xiong et al. 2013). Recently, Tomasi et al. (2013) demonstrated that Fe complexed by a water-extractable humic substance (WEHS) fraction can be utilized with higher efficiency than other natural Fe sources in tomato plants, even when supplied, as often occurs in the rhizosphere, in a mixture with other natural Fe complexes. This behavior has been attributed to the capability of the WEHS fraction to activate Fe uptake mechanisms and translocation also through a rapid co-regulation of different genes involved in Fe acquisition and its homeostasis (Aguirre et al. 2008; Buckhout et al. 2009; Zamboni et al. 2012). In addition, the fast activation of Fe acquisition mechanisms operating at the leaf level observed in plants fed with Fe-WEHS for 24 h could also, at least in part, contribute to the efficient use of Fe from this Fe source (Tomasi et al. 2009). As expected from these effects, Fe-deficient plants, when exposed up to 5 days to Fe-WEHS, exhibited a recovery of dry matter, Fe, and chlorophyll contents faster and with a greater extent than that observed by supplying other Fe sources (Pinton et al. 1999a).

In addition to the Fe supply, it has also been demonstrated that humic substances and in particular, WEHS fractions are able to stimulate per se active proton extrusion from roots and acidification of the surrounding environment (Pinton et al. 1997a, 1997b; Tomasi et al. 2013). As far as Fe nutrition is concerned, this effect could contribute to enhance the solubility of rhizospheric soil and apoplastic Fe (Cesco et al. 2002) and help in maintaining a more favorable apoplastic pH for the activity of the plasma membrane-Fe(III)-chelate reductase (Schmidt 1999); more generally, an enhanced release of protons might be related to the promotion of nutrient uptake which has been repeatedly observed after placing plants in contact with suitable concentrations of humic substances (Guminski et al. 1983; Maggioni et al. 1987; Pinton et al. 1999b; Varanini and Pinton 2001; Aguirre et al. 2008; Sudipta et al. 2009). Alterations of nutrient uptake and increased accumulation of cationic nutrients have been frequently observed in Fe-deficient tissues (Venkat Raju and Marschner 1972; Agnolon et al. 2002).

Despite the available literature on the acquisition, translocation, and leaf allocation processes of Fe in plants supplied with different Fe sources, limited information exists regarding the dynamics, at the leaf level, of the other elements during the recovery from the Fe-deficient symptoms. For this reason, time-dependent accumulation of nutrients and their distributions within leaf tissues of Fe-deficient cucumber plants treated with natural Fe sources like Fe-WEHS or Fe-citrate were studied using an innovative technique like synchrotron 2D-scanning μ-X-ray fluorescence (μ-XRF) analysis of leaf samples next to more conventional methods. Moreover, in order to evaluate the functionality of mechanisms operating at the root and leaf level, cation/anion uptake capacity of roots and leaf net CO2 assimilation activity were measured as well.

Experimental

Isolation and purification of WEHS and preparation of Fe-WEHS complex

WEHSs were obtained as reported by Tomasi et al. (2009). Briefly, WEHSs were extracted from finely ground sphagnum peat (2.5 g) by adding 50 mL of distilled water and shaking for 15 h at room temperature. Thereafter, the suspension was centrifuged at 12,000 g for 30 min, and the supernatant was filtered through a Whatman WCN 0.2-μm membrane filter. The resulting solution was acidified to pH 2.0 with H2SO4 and, in order to purify and concentrate the humified fraction, loaded onto an Amberlite XAD-8 column (Ø 20 mm and height 200 mm; Aiken et al. 1979). Adsorbed humic substances were washed with 100 mL of distilled water according to Aiken et al. (1979) and eluted from the column with 0.1 M NaOH. In order to remove the exchangeable metals, the solution was treated with Amberlite IR-120 (H+ form) up to pH 1–2 and then adjusted to neutrality with 0.1 M NaOH. The humified organic fraction was then freeze-dried before storage and dissolved in distilled water before use at a concentration of 167-mmol organic C per liter. As described by Tomasi et al. (2013), these humic substances are characterized by a low molecular weight and by a high content of oxygen-containing functional groups. Iron (Fe)-WEHS complex was prepared as described by Tomasi et al. (2013) by mixing WEHS fractions with FeCl3 in 5 mM Mes–NaOH at pH 6.0.

Plant material, growth conditions, and treatment of plants with Fe-WEHS or Fe-citrate

Cucumber seeds (Cucumis sativus L., cv. Serpente cinese, Dotto Sementi S.p.A., Italy) were germinated on filter paper moistened with 1 mM CaSO4. After 6 days, three seedlings were transferred for further 7 days to 350-mL glass vessels containing an aerated nutrient solution without Fe supply and grown in a growth chamber under controlled climatic conditions: day/night photoperiod, 16:8 h; radiation, 220 μEinsteins m−2 s−1; day/night temperature, 25:20 °C; relative humidity (RH), 70–80 %. The nutrient solution had the following composition (mM): K2SO4 0.7, KCl 0.1, Ca(NO3)2 2, MgSO4 0.5, and KH2PO4 0.1; (μM) H3BO3 10, MnSO4 0.5, ZnSO4 0.5, CuSO4 0.2, and (NH4)6Mo7O24 0.01 (Fe contamination is not higher than 10 ppb; Pinton et al. 1997a). The nutrient solutions were changed every 3 days and adjusted to pH 6.3 with 1 M NaOH. After 7 days of growing in Fe-free nutrient solution, 13-day-old cucumber plants showing visual deficiency symptoms (leaf chlorosis) were transferred for further 5 days to a nutrient solution with the same composition as reported above in the absence of any source of Fe (–Fe, control) or with the addition of Fe-WEHS or Fe-citrate. In order to reproduce conditions close to those occurring in the rhizosphere of alkaline soils where often Fe-deficiency symptoms in crops appear, the nutrient solution was buffered at pH 7.5 with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, and each Fe source was used at 1 μM final Fe concentration. The addition of 1 μM Fe as Fe-WEHS to the nutrient solution brought 5 mg Corg per liter of WEHS. Iron (Fe)-citrate was prepared according to von Wirén et al. (1994) by mixing an aliquot of citrate (10 % excess) with FeCl3. Iron concentration of the Fe sources was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES; VISTA MPX, Varian, Torino, Italy) before the addition to the nutrient solutions; furthermore, the photochemical reduction phenomena of the micronutrient complexed by the two ligands (WEHS or citrate) in the nutrient solution (Zancan et al. 2006) was limited to a minimum by covering all pots with black plastic foils during the entire experiment.

Analyses

During the 5 days of treatment, samples of plants were harvested daily; leaves were separated from stems and cotyledons and successively used for analytical determinations. The content of K, Ca, Mn, Cu, and Zn in leaf tissues was determined by ICP-AES, after digestion with concentrated HNO3.

The net CO2 assimilation by leaves and the rates of NO3 − and K+ net uptake by roots were measured using intact cucumber plants at the end of the first day of Fe-WEHS or Fe-citrate treatment. The net CO2 assimilation was determined using an infrared gas analyzer (Li-COR 6400) under the following conditions: 500-μmol s−1 air flux, 360 ppm CO2 concentration, and 400-μmol m−2 s−1 PAR. SPAD index values of fully expanded young leaves were determined using a portable SPAD-502 meter (Minolta, Osaka, Japan) after 5 days of treatments.

The net NO3 − uptake rates were measured as nitrate depletion from the uptake solution (Nikolic et al. 2012). Experimentally, after exposure to the different treatments, intact roots of two cucumber plants (about 0.4 g FW) were rinsed for 10 min in 1 mM CaSO4 and subsequently transferred to 20 mL of 200 μM KNO3 at pH 6.0 (uptake solution). Nitrate depletion from the uptake solution was measured over a 10-min period collecting 0.2 mL of the medium at 2-min intervals. Nitrate was determined spectrophotometrically at 410 nm according to Cataldo et al. (1975). The rates of net K+ uptake were determined by collecting aliquots (0.2 mL) of the uptake solution. Potassium content in the samples was determined by ICP-AES.

Synchrotron μ-XRF analyses were performed on fully expanded leaves after 5 days of treatment prepared as reported by Terzano et al. (2013). Briefly, leaf tissues were rinsed with deionized water, delicately dried with filter paper, and pressed between two plastic disks in order to keep the leaf flat; afterward, the “leaf sandwich” was frozen in liquid nitrogen and then freeze-dried under vacuum. At last, the intact freeze-dried samples were taped on a hollow flat aluminum sample holder for synchrotron analyses. From these samples, an area of 3 × 2 mm2, corresponding to the intersection between the midrib and a main lateral vein, was selected and analyzed by synchrotron 2D-scanning μ-XRF. The μ-XRF maps were collected in air at Beamline L at the DORIS III synchrotron of HASYLAB (DESY, Hamburg, Germany). The X-rays were focused to a 20-μm X-ray beam using an energy of 14 keV. The experimental setup consisted of a Ni/C multilayer monochromator, a polycapillary lens (working distance 5 mm, X-ray Optical System, Albany, USA), and a Vortex-EX (Radiant Detection Technologies) silicon drift detector. An Al filter was placed in front of the detector to reduce the intensity of the fluorescent radiation from low Z elements such as Ca and K, thus enhancing the relative intensity of the signals for higher Z trace elements, i.e., Fe and Br. The sample was moved in the X-ray beam using an XYZ motorized stage. The step size adopted was 20 μm, and the dwell time for each point was 1 s.

The XRF spectra, which represent the intensity of the fluorescent X-rays emitted by each element from the X-ray impinged area, were evaluated using the PyMCA software package (Sole et al. 2006) and in-house written routines. The intensity of the fluorescent X-rays emitted by each element gives an idea of the amount of emitting atoms present in the investigated portion of the leaf. In order to properly compare the spectra for different points in different samples, for each spectrum, the intensities of the peaks corresponding to the Kα emissions of the elements were divided by the intensity of the Kα emission of Br. This was used as an internal standard, since it is unlikely to be influenced by the different treatments (Tomasi et al. 2009). This ratio was calculated for Ca, K, Mn, Cu, and Zn. Also, element distribution maps were rescaled relatively to the integrated Br Kα signal of the whole scanned area in order to make them directly comparable. Total element concentrations for the different treatments were compared according to the intensity of the Kα emission for K, Ca, Cu, Zn, and Mn in the sum spectrum relatively to the intensity of the Br Kα line, used as an internal standard. The sum spectrum is the sum of all the spectra collected in the 3 × 2 mm2 investigated area (ca. 15,000 points).

Statistic analyses

Statistical validation (ANOVA Holm-Sidak; N = 3; P < 0.05) was performed using SigmaPlot 11.0 (Systat software, Point Richmond, USA).

Results

Figure 1 shows the dynamics of total accumulation (panel a) and concentration (panel b) of K and Ca in leaves of Fe-deficient plants after being supplied with 1 μM Fe as Fe-WEHS or Fe-citrate up to 5 days. As a control, a group of Fe-deficient plants was maintained for the same period in the absence of any Fe source. Plant exposure to the two Fe sources enhanced the total leaf accumulation of both elements and this behavior being particularly evident in plants treated with Fe-WEHS (Fig. 1a). On the other hand, the addition of the two Fe sources to the nutrient solution caused a sharp decrease in the foliar concentration of both elements that was more pronounced in Fe-WEHS-fed plants (Fig. 1b). Accumulation of micronutrients (Cu, Mn, and Zn) in the leaves is shown in Fig. 2a. The supply of Fe-WEHS favored the accumulation of these elements, particularly after 4 days of treatment. Fe-citrate did not modify the accumulation of these micronutrients, as their levels remained similar to those determined in Fe-deficient control plants. On the other hand, both Fe-citrate and Fe-WEHS, when added to the nutrient solution, induced a stronger decrease in the leaf concentration of these three micronutrients than in control plants (Fig. 2b). Leaf hydration measures showed that the Fe treatments did not modify the water content of these tissues (data not shown).

Fig. 1
figure 1

Total content (mg) of K and Ca in leaves of Fe-deficient cucumber plants treated up to 5 days with Fe-WEHS or Fe-citrate at a final Fe concentration of 1 μM or without any addition of Fe (–Fe, control) (a). Data of K and Ca concentration (mg/kg) are also reported (b). Data are means ± SD of three independent experiments with four replicates. Letters indicate a significative difference among the treatments for each time point (ANOVA Holm-Sidak; N = 3, P < 0.05)

Full size image
Fig. 2
figure 2

Total content (mg) of Cu, Mn, and Zn in leaves of three cucumber plants treated as reported in Fig. 1 (a). Data of Cu, Mn, and Zn concentration (mg Kg−1) are also reported (b). Statistics as in Fig. 1

Full size image

In order to investigate if supply of Fe-WEHS or Fe-citrate to Fe-deficient plants could alter the distribution of macronutrients and micronutrients, small areas of leaves were collected after the 5-day treatment with the two Fe sources and analyzed by means of synchrotron μ-XRF. Figure 3 displays the K, Ca, Mn, Cu, and Zn distribution maps. Concerning K, it is evident that in Fe-deficient plants, a very high accumulation occurred at the level of the main leaf veins. After the supply of Fe-WEHS or Fe-citrate, the extent of this phenomenon was much more limited: Interestingly, in the case of Fe-citrate, K was concentrated in both the main and secondary veins, while in the case of Fe-WEHS, principally in the midrib. The K concentration in the interveinal mesophyll areas did not appear to be affected by treatments. Calcium distribution maps revealed that this macronutrient was mainly located in the glandular trichomes. In this case, while Ca appeared to be distributed throughout the entire trichome of Fe-deficient leaves, in the Fe-citrate and Fe-WEHS fed plants, Ca was concentrated only at the base of the trichome. Also, Mn was accumulated at the base of the glandular trichomes in Fe-deficient leaves; however, after Fe-citrate or Fe-WEHS treatment, Mn was completely depleted from these locations. In Fe-deficient leaves, the Cu concentration was particularly high in the vascular system being the most intense in the mid part of the veins. Since in this plant species, xylem vessels are located between the phloem bundles, the Cu maps suggest that the micronutrient is concentrated in the xylem rather than in the phloem. The supply of Fe-citrate or Fe-WEHS determined a drastic decrease of Cu located at the vein level, although still maintaining higher levels than those recorded in the interveinal region. In particular, when considering the leaf veins, it is interesting to note that in Fe-citrate-fed plants, Cu appeared equally distributed in the midrib and in the lateral main vein, while in the Fe-WEHS-treated plants, a higher concentration was apparent only in the midrib. Similarly to Cu, although with a lower intensity, Zn also appeared to be concentrated at the vein level of Fe-deficient cucumber leaves and, more specifically, in the xylem vessels; in addition, in these tissues, microscopic intense Zn-rich spots throughout the main veins were visible. These were probably attributable to thin non-glandular trichomes, which, in cucumber plants, are mainly concentrated on the leaf veins (Choudhury and Copland 2003). In leaf maps of plants treated with Fe-citrate or Fe-WEHS, Zn was completely absent in the xylem, and the spots previously described in Fe-deficient leaves were still barely visible only in the Fe-citrate-fed plant.

Fig. 3
figure 3

Distribution of K, Ca, Mn, Cu, and Zn on a 3 × 2-mm2 area of a leaf imaged by 2D-scanning μ-XRF after 5 days of treatment. All the XRF intensities are calculated relatively to the signal of Br, used as an internal standard. Different images for the same element can be visually compared

Full size image

Table 1 reports the ratios calculated in the entire 3 × 2-mm2 analyzed areas (as shown in Fig. 3) using the fluorescent X-ray intensities emitted by each element (I) and the Kα emission of Br used as reference (I[Br Kα]). These ratios give an idea of the differences in element concentration (since the XRF intensity is directly proportional to the concentration) among the treatments. These differences can be better appreciated when considering these values in comparison with the –Fe leaves, regarded as 100 (numbers between brackets). For all the nutrients, results confirmed those presented in Figs. 1b and 2b, namely a marked reduction of all the element concentration in both the Fe-treated plants. With respect to the nutrient concentrations, as determined by ICP-AES after the digestion of the entire leaf tissues (Figs. 1b and 2b), with this approach, the extent of the decrease of nutrient concentration in leaves after the treatment with the two Fe sources was even more evident.

Table 1 Relative total concentration of K, Ca, Mn, Cu, and Zn as computed from synchrotron μ-XRF analyses on cucumber leaves after 5 days of treatment
Full size table

In order to evaluate if these modifications in the leaf nutrient content and their distribution in the tissues can be associated with a modification in the functionality of mechanisms operating at the root and leaf level and are related to the leaf nutrient allocation, cation/anion uptake capacity of roots and leaf net CO2 assimilation activity were measured after 1 day of Fe-citrate or Fe-WEHS supply. Figure 4a shows that the addition of 1 μM Fe as Fe-WEHS or Fe-citrate to the nutrient solution for 1 day caused a sudden increase of the root capacity to take up NO3 − anion, particularly in Fe-WEHS-fed plants. On the other hand, net K+ uptake rates were decreased by about 30 and 10 % by supplying Fe-citrate and Fe-WEHS, respectively, to Fe-deficient plants. Net CO2 assimilation activity at the leaf level was increased by the supply of Fe-citrate or Fe-WEHS (Fig. 4b); the increase was much higher when Fe-WEHS was used as Fe source.

Fig. 4
figure 4

Net uptake of nitrate and K (μmol g−1 root FW h−1) by roots of cucumber plants grown for 8 days in nutrient solution in the absence of Fe (a). Iron as Fe-WEHS complex or Fe-citrate was added to the solution at a final Fe concentration of 1 μM during the last day of nutrient solution culture. Data of net CO2 assimilation (μmol CO2 m−2 s−1) in leaves of three cucumber plants grown and treated as reported in Fig. 1 are also reported (b). Data are means ± SD of three independent experiments with four replicates. Letters indicate a significative difference among the treatments for each nutrient (ANOVA Holm-Sidak; N = 3, P < 0.05)

Full size image

Discussion

It is clearly demonstrated that the supply of Fe complexed by the WEHS fraction or citrate to Fe-deficient cucumber plants led to a recovery of Fe-deficiency symptoms with an extent strongly dependent on the type of the Fe source employed (Pinton et al. 1999a). In fact, plants supplied with Fe-WEHS exhibited a faster capacity to cope with this nutritional stress as evidenced by a quicker recovery of leaf Fe content and a concomitant sharp increase in the chlorophyll content (Fig. 5), as compared to plants treated with the other Fe source. In the present work, a marked recovery in the photosynthetic activity (net CO2 assimilation capacity of leaf) has been shown to be already evident after 1 day of treatment (Fig. 4). This evidence corroborates the idea of the effectiveness of the soluble Fe-humate complex, as a promptly utilizable Fe source (Chen et al. 2004; Pinton et al. 1999a; Cesco et al. 2002; Colombo et al. 2012). It has also been shown that the activation of Fe-acquisition mechanisms operating at the leaf level can contribute, at least in part, to the efficient use by plants of Fe from Fe-WEHS (Tomasi et al. 2009). Despite the vast literature concerning the dynamics and processes of Fe allocation in Fe-deficient plants after the supply with different Fe sources including also Fe-WEHS, little evidence exists regarding what happens at the leaf level to other nutrients during the recovery from Fe deficiency. Therefore, in the present work, the leaf allocation dynamics and the tissue distribution of major cationic macronutrient and micronutrients in leaves of Fe-deficient cucumber plants supplied with two different sources of Fe, Fe-WEHS, and Fe-citrate have been studied.

Fig. 5
figure 5

Fully expanded leaves of Fe-deficient cucumber plants treated for 5 days with Fe-WEHS or Fe-citrate at a final Fe concentration of 1 μM or without any addition of Fe (–Fe, control). SPAD index values are also given

Full size image

The results reported here show that the Fe supply via the two natural Fe sources to Fe-deficient cucumber plants, not only recovered foliar deficiency symptoms, like regreening and growth promotion (Fig. 5), but also lowered the nutrient (K, Ca, Cu, Mn, and Zn) concentrations in leaves (Fig. 1a, b). This phenomenon seems to be a component of the recovery process from the altered physiological metabolic condition of Fe-deficient plants. In fact, increased concentration of a number of cationic nutrients has been observed in tissues of Fe-deficient dicotyledonous plants; this effect has been related to an imbalance in the cation/anion uptake rates (Venkat Raju and Marschner 1972; Csog et al. 2011) to the activity of the Fe-deficiency-induced plasma membrane-Fe(III)-chelate reductase (Welch et al. 1993; Wulandari et al. 2014) and to the operation of Fe transporters which are able to transport also other metals, such as Cd, Cu, Mn, and Zn (Eide et al. 1996; Rogers et al. 2000). The depressed shoot dry matter production, which is characteristic in Fe deficiency, could surely also contribute to the altered concentration of cationic nutrients in leaves of Fe-deficient plants. In fact, the supply of Fe to deficient plants resulted in a fast recovery of the photosynthetic activity (Fig. 4b) restoring the dry matter accumulation capacity of plants (Fig. 5) with a consequent dilution effect for the nutrients. It is interesting to note that Fe-WEHS appears to be more efficient than Fe-citrate in lowering the cationic concentration (Figs. 1b and 2b); nonetheless, the nutrient contents in leaves of Fe-WEHS-fed plants (Figs. 1a and 2a) are the highest, demonstrating the quickest restart of leaf growth in these plants (Fig. 5). Considering also the greatest recovery of the carbon assimilatory activity in plants supplied with Fe-WEHS, these results indicate that the WEHS fraction does not act solely as an Fe chelate but that it is also able to interfere with the overall system of mineral nutrition. In this context, a lot of literature is available on the capability of soil humic substances to enhance per se nutrient accumulation in plant tissues (Rauthan and Schnitzer 1981; Chen and Aviad 1990; Varanini and Pinton 1995; García-Mina et al. 2004; Tomasi et al. 2013) affecting the mechanisms either at the root (Pinton et al. 1999a) or at the leaf level (Tomasi et al. 2009).

With respect to the root level, as previously observed in sunflowers and cucumber plants (Venkat Raju and Marschner 1972; Agnolon et al. 2002; Nikolic et al. 2007), Fe deficiency was able to depress the net nitrate uptake, while the absorption of K was increased. This might be due to a competition for carbon skeleton and reducing equivalent in the case of nitrate or in the case of K to the need for charge compensation at the membrane level following massive extrusion of protons. Moreover, nitrate reductase enzyme contains a heme Fe nucleus, and thus, in Fe deficiency, nitrate assimilation is slowed down (Alcaraz et al. 1986; Borlotti et al. 2012). Hence, the nitrate might accumulate in the plant tissue which induces a downregulation of its net uptake (Crawford and Glass 1998; Iacuzzo et al. 2011). Supplying Fe-WEHS or Fe-citrate to deficient plants, a sudden recovery of net nitrate uptake could be observed, while K uptake rates remained fairly high, especially in Fe-WEHS-treated plants (Fig. 4a). Since the two Fe sources were used at equimolar Fe concentration, the sole micronutrient concentration cannot explain the effect of Fe-WEHS supply. Notwithstanding the differences in experimental conditions used here in comparison to those employed in previous studies (Varanini and Pinton 1995), data of the present work are fairly consistent with an action of WEHS on nitrate uptake systems (Pinton et al. 1999b; Cacco et al. 2000), stimulation of proton-extruding mechanisms (Varanini et al. 1993; Pinton et al. 1997a; Tomasi et al. 2013), and enhancement of Fe-deficiency-induced reactions (Pinton et al. 1999a; Tomasi et al. 2013). Concerning the leaf, Tomasi et al. (2009) showed a fast activation of Fe-acquisition mechanisms operating at the leaf level in plants fed with Fe-WEHS already evident after 24 h from the onset of the root treatment with this Fe source. This evidence together with the results of this work supports the idea that the humic molecules are able to modify the dynamics of the accumulation of nutrients at the leaf level and also through actions exerted on the mechanisms responsible for the absorption and/or allocation of nutrients within plant tissues.

In this work, via synchrotron 2D-scanning μ-XRF analyses of leaf samples, the distribution of K, Ca, Cu, Mn, and Zn within the leaves has also been evaluated considering the effect caused by each of the two Fe sources when supplied to Fe-deficient cucumber plants. Results show that in leaves of Fe-deficient plants, K is concentrated at the level of the main vascular system, Ca is mainly located in the whole glandular trichomes, while Mn is accumulated only at the base of the trichomes. Copper accumulation is particularly high in the xylem vessels as well as Zn; moreover, the accumulation of the latter nutrient is particularly high also at the base of non-glandular trichomes at the vein level. It is interesting to note that K and Zn localization in the vascular region has been observed in plants grown in Cs-contaminated (Isaure et al. 2006) or Zn-enriched (Terzano et al. 2008) media, respectively. Calcium plays an important role in glandular trichomes exudation of tobacco leaves when plants are grown in elevated Zn or Cd levels (Sarret et al. 2013). Also, Zn, in high Zn-contamination conditions, was found to be concentrated in trichomes of tobacco (Sarret et al. 2006). Moreover, high concentration of Mn at the base of the trichomes has been observed in Zn- and Ni-hyperaccumulator plant species (Sarret et al. 2009; Broadhurst et al. 2004, 2009), whereas the high Cu concentration in the vascular cylinder of Cu-tolerant Commelina communis plants has been ascribed to the root-shoot Cu transport related to the metal tolerance of these plants (Shi et al. 2011). Considering these evidences, the cation distribution maps of Fe-deficient plants presented here suggest that the strategy implemented by cucumber plants to limit the consequences due to the increased content of these nutrients caused by Fe deficiency involves allocation processes of these elements very similar to those adopted by plants adapted to very high/toxic levels of metal availability. Furthermore, when Fe-deficient cucumber plants were supplied with Fe-WEHS or Fe-citrate, a partial redistribution to the remaining foliar blade of K, Cu, and Zn from the vascular region and of Ca, Zn, and Mn from trichomes occurred. This phenomenon appeared more evident in Fe-WEHS-fed plants suggesting that WEHS is not only able to stimulate the uptake and the allocation of nutrients at the leaf level but also favors their balanced distribution within the leaf tissue.

Conclusions

In conclusion, this work provides further evidence on the role of Fe-humate complexes in Fe nutrition of plants and supports the idea that the Fe-WEHS might effectively improve the recovery of Fe-deficiency symptoms favoring nutrient allocation at the leaf level concomitantly with their equilibrated distribution in the different leaf regions.

Moreover, the interrelations between nutrients occurring in leaves suggest that further research, including also those for the setup of agricultural practices focused at coping with Fe-deficiency problems in crops, should consider a multielement approach. This aspect is even more important when biofortification of crops and nutritional quality of food are concerned.