PHYSIOLOGIA PLANTARUM 122: 380–385. 2004 Printed in Denmark – all rights reserved
doi: 10.1111/j.1399-3054.2004.00405.x Copyright # Physiologia Plantarum 2004
Iron supply to tobacco plants through foliar application of iron citrate and ferric dimerum acid Victoria Ferna´ndeza,*, Gu¨nther Winkelmannb and Georg Eberta a
Fruit Science Department, Humboldt University of Berlin, Albrecht-Thaer-Weg 3, 14195 Berlin, Germany Microbiology & Biotechnology Department, University of Tu¨bingen, Auf der Morgenstelle 28, 72076 Tu¨bingen, Germany *Corresponding author, e-mail:
[email protected],
[email protected] b
Received 27 May 2004; revised 30 June 2004.
Experiments to assess the re-greening and the distribution of leaf-applied iron (Fe) within the aerial organs were developed with tobacco (Nicotiana tabacum L.) plants. Fe (III)-citrate and Fe-dimerum acid were applied to a part of the leaf and plant re-greening was monitored for 6 weeks after treatment. Fluid Fe increments associated with foliar Fe application were measured within 3 days after application. Fe distribution from the site of application within the leaf and the untreated above-ground plant parts was evaluated. Leaf fluid Fe concentration was determined by a
novel procedure based on Fe chelation by desferrioxamine E and high performance liquid chromatography separation on a reversed-phase column. The ferrioxamine E method enabled accurate determination of small amounts of Fe present in leaf fluid. Foliar Fe-dimerum acid and Fe (III)-citrate treatment to chlorotic tobacco plants induced new growth re-greening. It was concluded that the applied Fe-containing compounds penetrated the leaf, were distributed within the plant and that Fe supplied as both complexes could be utilized by the plant cell.
Introduction Iron (Fe) deficiency chlorosis is a common disorder affecting plants grown on calcareous, high pH soils. This abiotic stress can lead to serious yield and quality losses and requires the implementation of suitable plant Fe-deficiency correction strategies. The economic cost associated with Fe-deficiency correction under filed conditions is very high (Tagliavini and Rombola´ 2001). Fe spray application is a common method to cure Fe-deficiency chlorosis of tree, turf and field crops in areas where soil application of most Fe sources is ineffective (Mortvedt 1991). However, variable results after application of foliar Fe sprays to chlorotic plants have been described in the literature (Wallace and Lunt 1960, Abadı´ a et al. 2001). There is still limited knowledge concerning the factors involved in the process of leaf Fe penetration, plant translocation and cell Fe uptake which hinders the development of adequate foliar fertilization strategies to cure plant Fe deficiency.
All aerial plant organs are covered by a hydrophobic cuticle which is the principal barrier for the two-way exchange of water and solutes with the environment (Riederer and Schreiber 2001). Barthlott and Neinhuis (1997) observed a great microstructural diversity of leaf surfaces and a positive correlation between surface roughness, water-repellent properties and surface selfcleaning. As a consequence of atmospheric deposition, Burkhardt and Eiden (1994) proved the existence of water films on leaf surfaces. Cuticular hydration affects the number and size of aqueous pores and is an important factor concerning leaf solute penetration (Scho¨nherr 2001, 2002). The role of stomata in the process of foliar spray penetration is not fully understood (Ferna´ndez et al. 2003). However, stomatal infiltration may be an important pathway for the entry of leaf-applied chemicals (Eichert et al. 2002). Scho¨nherr and Bukovac (1972) showed that aqueous solutions with a surface tension higher than 30 mN m1 would fail to spontaneously
Abbreviations – Chl, chlorophyll; DFB, desferrioxamine B; DFE; desferrioxamine E; FoxB, ferrioxamine B; FoxE, ferrioxamine E; HPLC, high performance liquid chromatography; e, molar extinction coefficient.
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infiltrate open stomata. In contrast, Eichert and Burkhardt (2001) observed stomatal infiltration of aqueous solutions without the addition of surface active agents, but noted that normally less than 10% of stomata were involved in the penetration process. There is evidence that Fe deficiency impairs many plant physiological processes (Larbi et al. 2001). The role of the leaf apoplast concerning Fe delivery to the cell remains unclear (Kosegarten et al. 2001, Nikolic and Ro¨mheld 2002). Under Fe shortage conditions, micro-organisms and graminaceous species synthesize siderophores and phytosiderophores, which are low molecular weight, Fespecific compounds produced for the solubilization, transport and storage of Fe in the presence of other metal ions (Winkelmann and Drechsel 1997, Winkelmann 2001). The outstanding Fe chelating properties of many microbial siderophores make them suitable for therapeutic and analytical purposes (Gower et al. 1989, Kraemer and Breithaupt 1998). Working with cucumber and maize plants, Ho¨rdt et al. (2000) gained evidence of plant Fe utilization after root treatment with mono- and di-hydroxamate siderophores. This investigation was directed towards evaluating the distribution of leaf-applied Fe (III)-citrate and Fedimerum acid in aerial plant parts and the re-greening of new growth within 1 month after foliar treatment. Fe increments in plant fluids associated with foliar treatments were measured following a new HPLC-based Fe determination method (Ferna´ndez and Winkelmann 2005).
Materials and methods Plant culture Experiments were developed with 4–7 months-old Nicotiana tabacum var. ‘Virginia’ seedlings grown in sand culture. Plants were grown in the green-house at 25 C, 60–80% relative humidity and 16 h light/8 h dark photoperiod. Seedlings were watered daily with full-strength Arnon and Hoagland (1952) solution with (pH 5.5, 10.8 mM FeSO4 7H2O) or without Fe (pH 8 reached by addition of 10 mEq l1 NaHCO3). The nutrient solution contained: 1.02 g l1 KNO3, 0.492 g l1 Ca (NO3)2, 0.3 g l1 NH4H2PO4, 0.49 g l1 MgSO4 7H2O, 2.86 mg l1 H3BO3, 1.81 mg l1 MnCl2 4H2O, 0.08 mg l1 1 CuSO4 5H2O, 0.22 mg l ZnSO4 7H2O and 0.09 mg l1 H2MoO4 (MoO3 1 H2O). For foliar treatment plants were transferred from the greenhouse to an air-conditioned laboratory, at 20–22 C and 40–50% relative humidity. Fe solutions for foliar treatment 1, 3 and 5 mM Fe-dimerum acid and 1 and 15 mM Fecitrate (1 Fe : 20 citrate) solutions were applied to tobacco leaves. Purified ferric dimerum acid (purity . 99%) was isolated from Trichoderma viride as a degradation product of the main produced siderophores, Physiol. Plant. 122, 2004
coprogen and coprogen B (Anke et al. 1991). Purified desferrioxamine E (DFE) and ferrioxamine E (FoxE) were obtained from EMC microcollections GmbH, Tu¨bingen, Germany (Biophore Research Products, http://www.siderophores.info). Treatment solutions contained 1 g l1 Glucopon CSUP (Cognis Deutschland GmbH, Du¨sseldorf, Germany) and were applied at pH 5. Leaf treatment and sample collection Fe-containing solutions were applied to the distal part of the leaf including the tip, representing about 25% of the entire leaf surface. Foliar treatments were carried out by two different procedures: (1) 15 mM Fe (III)-citrate was applied by limiting the area of application with a 3.5 cm 3.5 cm transparent plastic foil stuck to the leaf tip with Tesa paper film (Tesa AG, Hamburg, Germany). The plastic piece covering the upper leaf surface was sealed with silicone (Bayer Healthcare AG, Leverkusen, Germany) and 50 ml of the Fe-containing solution were injected into the space between the plastic layer and the lower leaf side using a pipette. (2) 1 mM Fe (III)-citrate and 1, 3 and 5 mM Fe-dimerum acid solutions were applied with a brush over the upper and lower side part of the leaf tip, the amount of fluid used per leaf being recorded for further calculation (80–100 ml solution used per treated leaf). By the end of trials leaves were detached and all treated leaf surfaces were discarded. Surface Fe contamination of the untreated part of the receptor leaf was avoided by excising the treated area plus a margin of 0.5–1 cm. Fluid was obtained from the untreated part of the detached receptor leaf and the entire shoot tip. The same treatment solution was applied to two leaves per seedling. Six plants were used per treatment and each measurement was repeated twice. Samples were collected at 4, 44 and 68 h after application. six aerial plant parts and 8–16 leaves were centrifuged per treatment. Foliar treatments were applied in a chamber with Osram Lumilux Plus Eco, fluorescent tubes (approximately 1 m away from plants) at 25 C and 30–50% relative humidity. Plants were conditioned to light 2 to 3 h prior to foliar application. Fe-containing solutions were applied in the morning and seedlings were kept in the light for 24 h after treatment. Plants were well irrigated throughout the time span of the trial. Sample preparation Fluid was obtained by leaf centrifugation (Dannel et al. 1995, Lo´pez-Milla´n et al. 2001). Leaves were excised at the base of the rachis with a razor blade and were fixed on a strip of Parafilm M (American National CanTM, Chicago, IL), with the cut rachis remaining out of the plastic foil. The foil bearing the leaves was rolled and tightly secured with two or three, 0.03 m 0.01 m Parafilm strips. The end of the roll showing the excised leaf rachis was put into a 1.5-ml Eppendorf tube, the cap of which had been cut. The leaf roll partially inserted into 381
the Eppendorf tube was placed in a 50-ml plastic test tube, which was put into an open 250-ml centrifugation bottle. Leaf rolls were subsequently centrifuged at low speed (2500 g) for 15 min at 4 C (Sorvall RC-2 B Superspeed centrifuge; Kendro Laboratory Products GmbH, Langenselbold, Germany). The Eppendorf tubes containing leaf fluid were replaced by clean ones. The leaf rolls were further centrifuged at 4500 g and 4 C for 15 min. Eppendorf tubes containing the obtained fluids were stored at 4 C or 70 C.
Fe determination by the FoxE method Fe in plant fluids was determined after addition of DFE and HPLC separation on a reversed-phase column. Calibration curves were made by FoxE spectrophotometic reading (Ultrospec III, UV/VIS spectrophotometer; Pharmacia, Amersham Biosciences Europe GmbH, Freiburg, Germany) at 220 and 430 mn (lmax ¼ 430 mn, molar extinction coefficient (e) ¼ 2750 M1 cm1). Fe-saturated, FoxE solutions were measured on a Shimadzu HPLC System (Shimadzu, Duisburg, Germany) at 220 and 435 mn detector wavelengths. Samples including DFE and FoxE were separated on a reversed-phase Nucleosil 100 C18 column (20 250 mm, 7 mm; Grom Herrenberg, Germany) using a gradient of acetonitrile/water (6–40%) containing 0.1% trifluoroacetic acid (TFA) over 20 and 35 min (flow rate 1 ml min1). Prior to measurement, plant fluids plus DFE were incubated at 65 C for 30 min. The method was optimized by sample separation at 220 nm detector wavelength. However plant fluids were always measured at 435 nm detector wavelength. Contact to any Fe source was avoided at all stages and the HPLC system was thoroughly cleaned prior to sample analysis.
Results were statistically analysed using analysis of variance (ANOVA).
Results The FoxE method Fe in plant fluids was determined after addition of DFE and HPLC separation on a reversed-phase column (Ferna´ndez and Winkelmann 2005). The method was standardized by Fe determination via spectrophotometry and HPLC according to the area of the FoxE peak at 220 and 435 mn detector wavelengths (R2220nm ¼ 0.9985 and R2430nm ¼ 0.9974). Sample separation at 435 mn provided a sole peak corresponding to FoxE, the area of which relates to the Fe concentration of the probe (Fig. 1). A higher Fe concentration in the probe implies a larger area of the FoxE peak. Figure 1A relates to the amount of Fe present in the xylem fluid of Fe-sufficient tobacco leaves (4 mM FoxE). On the other hand, Fig 1B and C correspond to the separation of DFE plus apoplastic fluid obtained from the untreated part of receptor tobacco leaves, 68 and 44 h after foliar application of 15 mM Fe (III)-citrate (9 and 34 mM FoxE, respectively). Fluid Fe concentration after foliar treatment Fluid was obtained from the untreated part of the detached, receptor leaf and from the remaining, entire aerial plant parts. Application of 15 mM Fe-citrate to a part of a leaf induced an increment in the untreated part
mAbs 15 A
4 µM
10 5
FoxE
0 0
Leaf colour and chlorophyll Re-greening of chlorotic tobacco plants after foliar application of Fe-containing compounds was assessed with a Minolta CR 200 b photometer (Konica, Minolta Holdings Inc., Tokyo, Japan) on a weekly basis. The colour status of the six youngest leaves of each seedling was monitored for 6 weeks after treatment. Colour was measured following the L a* b* system and four samples were taken per leaf. A strong correlation between colour variable b and chlorophyll (Chl) a concentration was found for tobacco leaves (Y ¼ 0.003 X2 0.2437 X 1 5.6532, R2 ¼ 0.9687; where X is the colour variable b* and Y is the Chl a concentration). The Chl status of leaves was measured by the method of MacKinney (1941). Fresh tissue samples were frozen at 70 C and thereafter ground on a porcelain mortar. Chlorophylls were extracted in 80% acetone and Chl a and b concentrations were calculated after spectrophotometric reading (664 mn for Chl a and 647 mn for Chl b) (Ultrospec III, UV/VIS Spectrophotometer; Pharmacia). 382
5
10
15
mAbs 15 B
20 min 9.5 µM
10 5
FoxE
0 0
5
10
15
mAbs 15 C 10 5 0
20 min 34 µM
FoxE
0
5
10
15
20 min
Fig. 1. Chromatograms corresponding to tobacco leaf fluid plus DFE: (A) xylem fluid from a Fe-sufficient plant (4 mM FoxE) (B) apoplastic fluid obtained from the untreated part of receptor leaves 68 h after foliar treatment with 15 mM Fe (III)-citrate (9.5 mM FoxE) and (C) apoplastic fluid obtained from the untreated part of receptor leaves 44 h after foliar treatment with 15 mM Fe (III)-citrate (34 mM FoxE). Physiol. Plant. 122, 2004
2
not-treated part of receptor leaf
160
remaining aerial part plants 80
0
0
4
44
68
Time after treatment (h) Fig. 2. Fluid Fe concentration over time after foliar application of 15 mM Fe (III)-citrate to tobacco. Fluid was obtained from the untreated part of receptor leaves and from the remaining aerial plant parts excluding the treated leaves. Data are means SE (n ¼ 2).
of the receptor leaf and of the whole above-ground parts of the seedling (Fig. 2). Highest Fe concentrations were determined in samples collected 4 h after solution application. A decrease in fluid Fe concentration 44 and 68 h after treatment in comparison with fluid obtained 4 h after foliar Fe application was observed both for the untreated area of the receptor leaf and the remaining aerial part. Subsequently, maximum fluid Fe concentration associated with foliar treatment can be expected to occur between 4 and 44 h after treatment. Re-greening of chlorotic tobacco seedlings The treated area approximately corresponded to onequarter of the whole leaf area. Given the strong correlation found between Chl a and colour variable b*, colour measurements are expressed as Chl a concentration. Fe application to chlorotic tobacco leaves led to an increase in Chl a concentration of new growing leaves (Fig. 3). Maximum re-greening of new growth associated with Fe (III)-citrate application was achieved after 2 weeks. Fe-dimerum acid application induced a steady increment in Chl a concentration of new growth, the highest increase corresponding to 5 mM Fe treatment. Fe-treated chlorotic plants grew vigorously in contrast to untreated chlorotic plants. However, it must be noted that Fe treatment and especially 5 mM Fe-dimerum acid supply, induced early leaf senescence and in some cases yellowing of the treated leaf, probably indicating that solutions were still too concentrated. The present data provide indirect evidence of Fe translocation from the treated area to the growing tip.
Discussion In this investigation a new method to assess the distribution of leaf-applied, Fe-containing compounds and a novel procedure for Fe determination in plant tissues were introduced. This approach was attempted after Physiol. Plant. 122, 2004
Chlorophyll a (mg per 100 cm2)
Fe (µM )
240
1
0
Fe-deficient 1 mM Fe-dimerum a. 5 mM Fe-dimerum a. 0
Fe-citrate 3 mM Fe-dimerum a. Fe-sufficient
2 4 Time after treatment (weeks)
6
Fig. 3. Chlorophyll a increment of new growth after foliar application of Fe-containing compounds as compared to Fe-sufficient (continuous Fe supply via the root system) and Fe-deficient tobacco plants. The arrow indicates the time of foliar treatment. Data are means SE (n ¼ 12).
observing inconsistencies associated with Fe measurement of Fe-treated leaves. The aqueous chemistry of Fe and the insolubility of Fe hydrous oxide species in the absence of chelators have important biological and experimental implications. Foliar treatment with Fe (II)- and Fe (III)-salts has been avoided due to Fe chemistry constraints. Although Fe (II)-salt solutions rapidly oxidize under exposure to ambient air, Fe (III)-salt solutions readily form gelatinous hydrous oxide polymers once the pH is increased from 2 (Silver 1993). Subsequently, variable results can be obtained after spraying ionic Fe sources to Fe-deficient plants according to various factors such as local water pH. Thereby, it has been assumed that Fe salts are not good candidates for foliar fertilization purposes in spite of their low molecular weight and the ease with which Fe supplied as salts can penetrate the leaf under optimal conditions. Fe (III)-citrate and Fe-dimerum acid were selected for foliar treatment, as they were found to be easily translocated within the plant in comparison with other Fe-containing compounds (Ferna´ndez 2004). Working with maize and cucumber, Ho¨rdt et al. (2000) showed that plants could utilize Fe supplied as dimerum acid and fusarinin complexes. However and prior to this report, there was no information available concerning the penetration and distribution of leaf-applied ironsiderophores. Estimating foliar penetration of Fe-containing compounds proves complicated due to Fe chemistry constraints and to the risk of Fe contamination of the treated leaf surface. Subsequently, an alternative means to evaluate the distribution of leaf-applied Fe was developed in this investigation. Fe was recovered in fluid obtained from untreated plant parts, some hours after foliar treatment with Fecontaining compounds. The mode of foliar treatment and 383
sample collection used in this study was aimed at minimizing the risk of probe Fe contamination. Searching for a reliable method to determine exogenous Fe in plant fluids, a new HPLC-based method was introduced. The procedure is similar to the one described by Gower et al. (1989) or Kraemer and Breithaupt (1998), in which Fe was measured by HPLC after addition of desferrioxamine B (DFB) to organic fluids. However, DFE has a higher affinity for Fe13 in comparison with DFB [1032.5 versus 1030.5 for FoxE and ferrioxamine B (FoxB), respectively] (Boukhalfa and Crumbliss 2002). Another advantage of the FoxE method is that DFE does not interact with compounds present in samples in contrast to the basic character of the DFB molecule (Keberle 1964). DFE can extract Fe from a variety of Fe-complexes and insoluble Fe compounds, the FoxE molecule being stable down to pH 1. The FoxE method enabled accurate determination of small and larger amounts of Fe present in plant fluids (Ferna´ndez and Winkelmann 2005). For optimal complex formation, fluid samples containing DFE were incubated for 30 min at 65 C. To avoid interference with compounds present in plant fluids, probes were always measured at 435 mn detector wavelength. The only peak appearing in chromatograms after sample separation at 435 mn, is the one corresponding to FoxE. Subsequently, Fe determination by the FoxE method proves accurate and simple to develop. To take account of the importance of light concerning the penetration, distribution and cell uptake of Fe-containing compounds, foliar treatments were always given in the light. To achieve good leaf wetting and low surface tension below 30 mN m1, solutions contained 1 g l1 Glucopon CSUP (Scho¨nherr 2001). The solutions were applied at pH 5 to avoid alteration of the ion exchange capacity of the leaf surface (Scho¨nherr and Hu¨ber 1977, Chamel 1996). Maximum fluid Fe concentration after 15 mM Fecitrate application was recovered 4 h after treatment. Fluid Fe levels 44 and 68 h after treatment steadily decreased over time. The reduction in xylem and apoplastic fluid Fe concentration some time after treatment may indicate that Fe was translocated to the root and/or that it entered the symplast. Aware of the reduction in fluid Fe concentration over time following the application of Fe-containing compounds, assessment of fluid Fe concentration 1 month after treatment was not estimated relevant. However, indirect evidence of Fe-citrate and Fe-dimerum acid penetration was gained via a physiological response in plants, namely the re-greening of new growth. Following foliar treatment, the Fe increase in fluid obtained from above-ground plant parts was much lower than the Fe concentration measured for the untreated part of detached, receptor leaves. Similarly, the decrease in Fe concentration over time was less pronounced in fluid obtained from the aerial parts of tobacco seedlings as compared to the untreated part of receptor leaves. Results show that a certain proportion 384
of the leaf-applied Fe was distributed within the treated leaf and translocated to the shoot tip. The obtained data are in agreement with the findings of Brown et al. (1965) and Basiouny and Biggs (1971) concerning the translocation of leaf-applied Fe to actively growing regions of kidney bean, sorghum, cotton and citrus plants. The obtained data suggest that maximum Fe distribution within the treated leaf can be expected to occur between 4 and 44 h after foliar treatment. Leaf application of Fe-citrate and Fe-dimerum acid to chlorotic tobacco plants led to an increase in Chl a concentration of growing tissues. Plants treated with Fe-dimerum acid re-greened over time, and the highest Chl a levels associated with Fe (III)-citrate application were observed 2 weeks after the beginning of the trial. Results indicate a more rapid physiological response to Fe (III)-citrate in comparison with the effect of Fe-dimerum acid, which induced maximum re-greening 6 weeks after treatment. It was concluded that cells were able to utilize Fe supplied as Fe (III)-citrate and Fe-dimerum acid, as derived from the increased Chl a concentration of new leaves, which reached the level of Fe-sufficient tobacco plants. Optimal new growth greening was attained after 5 mM Fe-dimerum acid application, treatment which also hastened mature leaf senescence. Early leaf senescence was possibly a result of an excessively high Fe concentration of the leaf-applied solution. The capacity of Fe to cause oxidative damage to the plant should be taken into consideration. Significant greening was also attained after treatment with 1 mM Fe-dimerum acid. Variable Fe-chlorotic plant physiological responses to different foliar Fe spray formulations and concentrations have often been described in the literature (Leonard 1967, Rombola´ et al. 2000, Abadı´ a et al. 2002). In this investigation evidence of Fe (III)-citrate and Fe-dimerum acid leaf penetration and exogenous distribution within the tobacco plant was gained. Earlier results have shown that mono- and dihydroxamates resulting from microbially degraded hexadentate complexes such as coprogen, produced by Penicillium species, can be used as iron sources at the root surface of plants (Ho¨rdt et al. 2000). It is interesting to note that in this study another soil fungus, Trichoderma viride, is a producer of coprogen and dimerum acid (Anke et al. 1991). Thus, iron availability to plants is enhanced after degradation of hexadentate hydroxamates to tetra- and bidentate hydroxamate iron complexes. The mechanism of iron release to the plant cell is still unknown. Tetradentate hydroxamates such as Fe-dimerum acid show a slow ligand exchange kinetics requiring very acidic conditions and do not significantly differ from hexadentate complexes. However, redox-facilitated ligand exchange is easier to perform in comparison with hexadentate hydroxamate complexes (Boukhalfa and Crumbliss 2002). We therefore assume that in addition to the excellent transport properties of tetradentate hydroxamate iron complexes, these compounds are better substrates for reductive iron release at the plant cell, Physiol. Plant. 122, 2004
due to their lower redox potentials [359 mV versus NernstHydrogen-Electrode (NHE)] as compared to hexadentate complexes (coprogen 440 mV versus NHE). Results suggest that rate of Fe foliar penetration, translocation and the subsequent physiological response may vary according to different Fe-carriers and Fe concentrations. However, the significance of the factors involved in the process of leaf Fe penetration and Fe distribution within the plant is not fully understood and more research is required to improve the efficiency of Fe foliar sprays as a successful strategy to cure plant Fe deficiency.
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Edited by J. K. Schjørring Physiol. Plant. 122, 2004
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