Iron Induces Root and Leaf Ferric Chelate Reduction Activity in Grapevine Rootstock 140 Ruggeri

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  • 1 Department of Horticultural and Crop Science, Agricultural University of Athens, Iera Odos 75, 118 55, Athens, Greece

Ferric chelate reduction (FCR) activity of roots and leaves was determined in the grapevine rootstock 140 Ruggeri under iron (Fe)-deficient and Fe-sufficient conditions. Micropropagated plants were subjected to 0 or 40 μm Fe(III) EDTA in hydroponic culture. After 10 days of treatments, Fe-deficient plants had a lower level of root FCR activity and chlorophyll content compared with Fe-sufficient plants. Iron supply (20 μm) to Fe-deficient plants caused a rapid increase of root FCR activity (five times higher than initial values) and plants restored leaf chlorophyll content, whereas those not supplied with Fe retained reducing activity at low levels. In Fe-sufficient plants, root FCR activity decreased gradually when Fe was removed from the nutrient solution, reaching the same low levels as the Fe-deficient plants. Iron-sufficient plants displayed constitutively elevated root-reducing capacity for 3 weeks in culture under Fe-sufficient conditions. In Fe-deficient plants, root FCR activity was confined at the apical region of the lateral roots, whereas in Fe-treated plants, activity was detected along almost the whole length of the lateral roots. Root hair development, from the aspect of length and density, proved to be independent of the Fe status of rootstock plants. Leaf FCR activity was measured in mesophyll protoplasts from in vitro-growing Fe-deficient and Fe-sufficient plants. Mesophyll protoplasts reducing activity in Fe-deficient plants appeared to be decreased 43.5% or 40.3%, on a protoplast number or protein basis, respectively, in comparison with that in protoplasts from Fe-sufficient plant leaves. The results suggest that Fe is one of the agents inducing root and leaf FCR activity in grapevine rootstock 140 Ruggeri, altering furthermore its localization pattern in roots.

Abstract

Ferric chelate reduction (FCR) activity of roots and leaves was determined in the grapevine rootstock 140 Ruggeri under iron (Fe)-deficient and Fe-sufficient conditions. Micropropagated plants were subjected to 0 or 40 μm Fe(III) EDTA in hydroponic culture. After 10 days of treatments, Fe-deficient plants had a lower level of root FCR activity and chlorophyll content compared with Fe-sufficient plants. Iron supply (20 μm) to Fe-deficient plants caused a rapid increase of root FCR activity (five times higher than initial values) and plants restored leaf chlorophyll content, whereas those not supplied with Fe retained reducing activity at low levels. In Fe-sufficient plants, root FCR activity decreased gradually when Fe was removed from the nutrient solution, reaching the same low levels as the Fe-deficient plants. Iron-sufficient plants displayed constitutively elevated root-reducing capacity for 3 weeks in culture under Fe-sufficient conditions. In Fe-deficient plants, root FCR activity was confined at the apical region of the lateral roots, whereas in Fe-treated plants, activity was detected along almost the whole length of the lateral roots. Root hair development, from the aspect of length and density, proved to be independent of the Fe status of rootstock plants. Leaf FCR activity was measured in mesophyll protoplasts from in vitro-growing Fe-deficient and Fe-sufficient plants. Mesophyll protoplasts reducing activity in Fe-deficient plants appeared to be decreased 43.5% or 40.3%, on a protoplast number or protein basis, respectively, in comparison with that in protoplasts from Fe-sufficient plant leaves. The results suggest that Fe is one of the agents inducing root and leaf FCR activity in grapevine rootstock 140 Ruggeri, altering furthermore its localization pattern in roots.

Iron (Fe) is an essential nutrient for plants, which catalyzes crucial cellular functions such as chlorophyll synthesis, chloroplast development, and antioxidative cell protection (Marschner, 1995). Despite being abundant in soils, Fe mainly exists as the insoluble, nonavailable to plants, ferric [Fe(III)] form (Lucena, 2000). Plants have developed two separate strategies to acquire Fe(III) from soils. In strategy I plants (dicots and nongraminaceous monocots), a reduction step of Fe(III) to the bioavailable ferrous form [Fe(II)] is required before the transmembrane import of iron (Marschner and Römheld, 1994). In strategy II plants (graminaceous species), soil Fe(III) is chelated and transferred by the plant-exudated phytosiderophores without being previously reduced (Marschner and Römheld, 1994). However, the identification of common elements in both strategies has weakened the strict separation between them (Schmidt, 2003).

Iron deficiency induces ferric chelate reduction (FCR) activity in roots in strategy I plant species in conjunction or not with other adaptive responses such as rhizosphere acidification and changes in root hair and transfer cell development (Schmidt, 1999). Nevertheless, in some annual and perennial plants, root FCR activity is induced at the presence of Fe, whereas under deficiency conditions, reduction activity remains at low levels (Bohórquez et al., 2001; Gogorcena et al., 2000, 2004; Ojeda et al., 2003; Zouari et al., 2001).

The photosynthetically active mesophyll cells, and specifically their chloroplasts, are one of the major sinks of Fe in plants (Imsande, 1998; Marschner, 1995). Iron is allocated throughout the plant as Fe(III) chelates, which must be reduced to Fe(II) to enable its transportation in the mesophyll cells (Hell and Stephan, 2003). Ferric reduction capacity of leaf cells has been until now estimated in a few plant species with the use of intact leaves (Kosegarten et al., 1999), excised leaf pieces (Larbi et al., 2001; Nikolic and Römheld, 1999), plasma membrane preparations (Brüggemann et al., 1993; González-Vallejo et al., 1999), and leaf protoplasts (González-Vallejo et al., 2000).

Iron deficiency is a widespread nutritional disorder among the cultivated plant species and particularly in grapevine, which is induced in calcareous and alkaline soils and is known as Fe chlorosis (Mengel, 1995). Impaired Fe nutrition causes severe symptoms in grapevine such as chlorotic leaves and decreased growth and fruit yield (Bavaresco et al., 2003; Bavaresco and Poni, 2003). A wide range of resistance degrees to induced Fe deficiency can be tracked among grapevine species (Bavaresco et al., 1994). Vitis vinifera cultivars grown by their own roots are generally efficient regarding Fe acquisition under Fe deficiency conditions induced in calcareous soils (Galet, 1979). On the contrary, grapevine rootstocks, which are interspecific hybrids used for the control of phylloxera, exhibit diverse behavior under equivalent conditions (Tagliavini and Rombolá, 2001), and the utilization of the most tolerant ones offers the most efficient way for alleviating Fe chlorosis in grapevine. Rootstock genotype affects to a great extent Fe nutrition in the scion cultivars whenever Fe availability is limited as a result of soil conditions (Bavaresco et al., 1993, 2000).

Grapevine is considered to be a strategy I plant because root Fe(III)-reducing capacity and rhizosphere acidification have been demonstrated for several rootstocks and vinifera cultivars under Fe deficiency conditions (Bavaresco et al., 1991; Brancadoro et al., 1995; Jiménez et al., 2007; Nikolic et al., 2000). However, there is insufficient knowledge about the specific mechanisms that are used by grapevine rootstocks for Fe acquisition and plant responses to external Fe supply. Root FCR activity has been studied with time course experiments in the susceptible to Fe deficiency rootstock V. riparia and the tolerant vinifera cultivar ‘Cabernet Sauvignon’ (Jiménez et al., 2007). Furthermore, localization of root FCR activity and contribution of root hairs to Fe acquisition processes have not yet been studied in grapevine. Recently, leaf ferric reduction activity was determined with the use of leaf disks in Concord grapevine (Smith and Cheng, 2007).

The aim of this work was to study the characteristics of FCR activity in roots and leaves of the grapevine rootstock 140 Ruggeri, which is highly tolerant to Fe deficiency stress conditions (Tagliavini and Rombolá, 2001). Root FCR activity was studied in time course experiments after addition or removal of Fe from the nutrient solution, in Fe-deficient and Fe-sufficient micropropagated rootstock plants, respectively. Localization of FCR activity in roots and the contribution of root hairs to Fe(III) acquisition were studied. In parallel, leaf FCR activity was determined with the use of mesophyll protoplasts from in vitro-growing Fe-deficient and Fe-sufficient 140 Ruggeri rootstock plants.

Materials and Methods

Plant material, culture, and experimental design.

Micropropagated plants of grapevine rootstock 140 Ruggeri (V. berlandieri × V. rupestris) were used. One-node stem explants from in vitro-growing mother plants were cultured in auxin-free culture medium (Roubelakis-Angelakis and Zivanovitc, 1991). Once the plants had developed approximately four expanded leaves and a well-grown root system (≈0.4 g fresh weight), they were removed from in vitro culture and acclimatized under ex vitro conditions in hydroponic culture with half-strength Hoagland's solution, pH 5.8, supplemented with 20 μm Fe(III) EDTA. By this procedure, a homogenous population of rootstock plants was obtained. After 1 week, acclimatization was completed and plants were used in the experiments. Throughout the study, Fe(III) EDTA was used as the Fe source.

Before treatments, rootstock plants were transferred in half-strength Hoagland's solution, pH 5.8, with either 0 or 40 μm Fe for 10 d to develop Fe-deficient and Fe-sufficient plant populations, respectively (200 plants per population). By the end of this period, Fe-deficient plants had developed Fe chlorosis symptoms. After this pretreatment period, each of these plant populations (Fe-deficient and Fe-sufficient plants) was divided into two groups of plants (100 plants per group), which were then transferred in fresh half-strength Hoagland's solution and subjected to Fe treatments, 0 or 20 μm Fe. The pH was set to 5.8 with the use of NaOH and nutrient solution was renewed every week. During the week, pH of the nutrient solution was checked but not adjusted to the initial value. Plants were cultured in black plastic containers with 0.5 L nutrient solution. Two plants were placed in each container. The solution in the containers was continuously aerated with air pumping. All nutrient solutions were prepared with distilled water.

Iron-deficient and Fe-sufficient plants grown under in vitro conditions were used for mesophyll protoplast isolation. To obtain these plants, newly rooted one-node explants were removed from the initial rhizogenesis culture medium and transferred to a new one supplemented with either 0 or 20 μm Fe. This medium did not contain sucrose and the pH was 5.8. The concentration of the agar (0.7%, w/v) in both media allowed the removal and transfer of the plants without causing any damage to the roots.

Plant micropropagation, culture, acclimatization, and experiments were held under a controlled environment in growth chambers. Temperature was 25.5 °C, relative humidity 75%, and photoperiod 16/8 h light/dark with a photosynthetic photon flux of 300 μmol·m−2·s−1 provided by cool white fluorescent lamps. Acclimatization was achieved under the same conditions except that relative humidity was 100% at the beginning continuously descending to 75% by the end of the procedure.

The experiments were performed in a completely randomized design with four replications per treatment. Significant differences between means were determined with the use of t test. Regression analysis was also carried out to test linearity in FCR activity assays.

Preparation of mesophyll protoplasts.

Protoplasts were isolated according to Katsirdakis and Roubelakis-Angelakis (1992) with modifications. The upper fully expanded leaves from Fe-deficient and Fe-sufficient plants were used after 12 d of treatment. Leaf pieces (1 × 0.2 cm) were placed on incubation medium consisting of 0.7 M sucrose, 10 mm 2-(N-Morpholino)ethanesulfonic acid (MES)-NaOH pH 5.5, 1 mm CaCl2, and the cell wall digesting enzymes cellulase R-10 (1%, w/v) and macerozyme R-10 (0.5%, w/v) (Duchefa Biochemie, Haarlem, The Netherlands). Incubation was carried out in 4 h in the dark and at a temperature of 25.5 °C. After digestion, mesophyll protoplasts were released in the incubation medium with a gentle shaking. Protoplast suspension was loaded in centrifuge tubes and overlayered with the isolation medium consisting of 0.7 M mannitol, 10 mm MES-NaOH pH 5.5, and 0.5 mm CaSO4. Protoplasts were isolated after centrifugation at 1100 rpm for 15 min (SS-34, model Evolution RC; Sorvall, Kendro Lab. Products, Asheville, NC) from the interface between the incubation and the isolation medium. Isolated protoplasts were washed three times with isolation medium and were immediately used in reduction assays. The number of intact protoplasts per unit volume was determined before the assays, in a microscope with a hemocytometer, on the basis of Evans blue stain exclusion.

Ferric chelate reduction assays.

Ferric chelate reduction activities were quantified with the use of 3-(2-pyridyl)-5, 6-diphenyl-1, 2, 4-triazine sulfonate (ferrozine), which chelates the reduced Fe from Fe(III) EDTA and forms the stable, water-soluble, and no permeable [Fe(II)-ferrozine] complex (Schmidt et al., 2000).

Root FCR activity was determined in intact plant roots and in excised root segments. Two different root parts were excised, the root apical and subapical segments (both 1.5 cm long). Intact and excised roots were rinsed with distilled water to remove nutrients. Then they were soaked in 1 mm EDTA for 5 min to eliminate apoplastic Fe and washed three times with distilled water to reduce excess EDTA (Ojeda et al., 2004). Ferric chelate reduction assays were conducted as has been previously described (Vizzotto et al., 1997) with modifications. The assay's solution consisted of 10 mm MES-NaOH pH 5.5, 0.5 mm CaSO4, 100 μm Fe(III) EDTA, and 300 μm ferrozine. Quantitative determination of root FCR activity in intact plants (eight plants per treatment) was performed by immersing the roots of a single plant in 50 mL of assay solution. The absorbance was recorded at various time intervals since the initiation of the assay. Plants were used once in FCR assays, root fresh weight was recorded, and then discarded from the experiments. Excised roots samples, ≈0.1 g each, were immersed in 2 mL of assay solution. The assay solution for the determination of mesophyll protoplasts’ FCR activity comprised the isolation medium, 100 μm Fe(III) EDTA, and 300 μm ferrozine. Each protoplast assay used 5 × 104 protoplasts suspended in 1 mL solution. Protoplasts were removed from the assay solution with centrifugation, and the supernatant was used for the absorbance measurements. All reduction assays were performed in the dark, at 25.5 °C, under continuous stirring. Absorbance of the ferrozine complex was recorded at 562 nm with a double-beam spectrophotometer (model U-2001; Hitachi, Tokyo). Reduced Fe [Fe(II)] was calculated with the use of an extinction coefficient of 25,200 M−1·cm−1. Control assays were also conducted to correct any unspecific chelation of ferrozine.

Localization of root FCR activity was determined by embedding the entire root system of the plants (12 plants per treatment), 7 d after treatments were imposed, in a medium containing 0.5 mm CaSO4, 0.5 mm Fe(III)EDTA, 0.5 mm ferrozine, and 0.7% (w/v) agar (Schmidt et al., 2000). Activity staining was developed after 1 h in the dark and 25.5 °C and plates were photographed.

Chlorophyll and protein measurements.

Leaf chlorophyll index was determined nondestructively using a portable chlorophyll meter (SPAD-502; Minolta, Ramsey, NJ). SPAD values, of the first fully expanded leaf in Fe-deficient and Fe-sufficient grapevine rootstock 140 Ruggeri plants treated with either 0 or 20 μm Fe (eight plants per treatment), were determined at the beginning of the treatments (0 d) and after 7 d. Protoplast protein extraction was carried out as described (Siminis et al., 1994) and protein concentrations were quantified according to Lowry et al. (1951). The total chlorophyll content of protoplasts was determined after extraction with 96% ethanol applying the equations of Wintermans and De Mots (1965).

Microscopy.

Observations of root hair development and mesophyll protoplasts were performed with a light microscope (Olympus BX 40, Tokyo). Root regions either exhibiting or not ferric chelate reduction activity were marked and examined under the microscope for root hair development 7 d after treatments were imposed. Root hair patterns were recorded on positive film.

Results

After a 10-d pretreatment period, Fe-deficient plants showed low root FCR activity in contrast with Fe-sufficient plants, which exhibited significant higher values (Fig. 1). Time course measurements during the assays revealed that root FCR activity was linear for at least 2 h in both Fe-deficient and Fe-sufficient plant populations (Fig. 1). On the basis of the previous results, all root FCR activity measurements were carried out in assays with 1-h incubation.

Fig. 1.
Fig. 1.

Ferric chelate reduction activity of intact roots in iron (Fe)-deficient (○) and Fe-sufficient (•) grapevine rootstock 140 Ruggeri plants. Rootstock plants were cultured in nutrient solution with either 0 or 40 μm Fe for 10 d. Assays were run for 2 h and reduction activity was determined at 30-min intervals. Data are means ± se of three different experiments with n = 8. Lines represent linear regression analysis of the means and are different for P ≤ 0.0001 (Fe-deficient, y = –2.5 + 1.557x, slope = 1.557 ± 0.061, r 2 = 0.99; Fe-sufficient, y = 2 + 4.773x, slope = 4.773 ± 0.373, r 2 = 0.96).

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

Root FCR activity changes were recorded in Fe-deficient and Fe-sufficient plants cultivated in nutrient solution with either 0 or 20 μm Fe (Figs. 2 and 3). Iron-deficient plants, resupplied with 20 μm Fe, exhibited a rapid increase of root FCR activity (Fig. 2). Maximum values were observed 3 d after Fe addition, whereas elicited root FCR activity was five times higher than the initial one. Deficient plants not supplied with Fe retained root FCR at low levels (Fig. 2). Root FCR activity in Fe-sufficient plants decreased gradually when Fe was removed from the nutrient solution (Fig. 3). The lowest values were recorded 4 d after the initiation of the treatment. Iron-sufficient plants retained the elevated root reduction capacity during cultivation with 20 μm Fe (Fig. 3). Root FCR activity in Fe-deficient plants resupplied with Fe reached Fe-sufficient plants’ FCR values 4 d after Fe supply (Figs. 2 and 3).

Fig. 2.
Fig. 2.

Changes in root ferric chelate reduction activity of iron (Fe)-deficient grapevine rootstock 140 Ruggeri plants after Fe supply. Reduction activity was determined in intact roots of plants in nutrient solution supplied either with 0 (○) or 20 (•) μm Fe. Measurements were conducted daily throughout 1 week after treatments were imposed. Data are means ±se of three different experiments with n = 8.

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

Fig. 3.
Fig. 3.

Changes in root ferric chelate reduction activity of iron (Fe)-sufficient grapevine rootstock 140 Ruggeri plants after Fe removal. Reduction activity was determined in intact roots of plants in nutrient solution supplied either with 0 (○) or 20 (•) μm Fe. Measurements were conducted daily throughout 1 week after treatments were imposed. Data are means ±se of three different experiments with n = 8.

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

Both Fe-deficient and Fe-sufficient grapevine rootstock plants retained a high root FCR activity when grown at the presence of Fe for an additional 2-week period under the same experimental conditions (Fig. 4). On the contrary, the absence of Fe resulted in low FCR activity in both plant populations for the same growth period. Iron-deficient plants growing with 0 μm Fe presented a slightly higher root FCR activity after 14 d than after 7 d since the initiation of the experiment (Figs. 2 and 4), possibly as a result of new lateral root formation.

Fig. 4.
Fig. 4.

Root ferric chelate reduction activity of grapevine rootstock 140 Ruggeri plants growing in nutrient solution with either 0 or 20 μm iron (Fe). Reduction activity was determined in intact roots 14 and 21 d after treatments were imposed to Fe-deficient and Fe-sufficient plants. Data are means ±se of three different experiments with n = 8. Means were separated with t test and * and ** denote significant differences at P ≤ 0.05 and P ≤ 0.01, respectively.

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

SPAD values were determined in the first fully expanded leaf of plants in all treatments and were found to be higher in Fe-sufficient plants (Table 1). Moreover, as the increased SPAD values indicate, Fe-deficient rootstock plants restored chlorophyll content when supplied with Fe compared with nontreated Fe plants. Removal of Fe from the nutrient solution caused a significant decrease in SPAD values of Fe-sufficient plants. SPAD values presented in Table 1 correspond to chlorophyll concentrations from 1.33 to 2.10 μg of total chlorophyll per milligram leaf fresh weight (data not shown).

Table 1.

SPAD values of the first fully expanded leaf in iron (Fe)-deficient and Fe-sufficient grapevine rootstock 140 Ruggeri plants treated with either 0 or 20 μm Fe.z

Table 1.

Iron supply caused a significant shift in the localization pattern of FCR activity in roots. In Fe-deficient plants, root FCR activity was localized at the apical region of the lateral roots, whereas in Fe-sufficient plants, activity was detected along almost the total length of the lateral roots (Fig. 5). Color intensity in agar-embedded roots indicates the level of FCR. Excised apical and subapical root parts, both 1.5 cm long, were used for the quantitative determination of FCR activity along the roots (Fig. 6). In Fe-deficient plants, almost the entire determined FCR activity was located at the apical 1.5-cm long region of the lateral roots, whereas in Fe-sufficient plants, the bulk of the activity was found at subapical regions. In Fe-deficient plants, root FCR values determined with the 0- to 1.5-cm apical segments were ≈5 times higher than those measured with intact roots (Figs. 2 and 6). Meristematic parts of the roots (root tips) did not exhibit any reduction activity (Figs. 5 and 7).

Fig. 5.
Fig. 5.

Localization of ferric chelate reduction activity along the lateral roots of grapevine rootstock 140 Ruggeri plants. Experiments were performed in excised roots from plants, treated with either 0 or 20 μm iron (Fe), 7 d after plants were subjected to treatments. Visualization of reduction activity was recorded by embedding the roots in agar medium supplemented with Fe(III) EDTA and ferrozine. Color intensity indicates the degree of reduction activity. The experiment was performed four times with similar results. Bar = 10 mm.

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

Fig. 6.
Fig. 6.

Ferric chelate reduction activity along the lateral roots of grapevine rootstock 140 Ruggeri plants. Data were obtained with excised roots 7 d after iron (Fe) treatments, 0 or 20 μm Fe, were imposed to Fe-deficient plants. The apical (0 to –1.5 cm) and subapical segment (1.5 to 3 cm), both 1.5 cm long, were excised and reduction activity was determined separately. Data are means ±se of two experiments with four replications each. Means were separated with t test and ** and *** denote significant differences at P ≤ 0.01 and P ≤ 0.001, respectively.

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

Fig. 7.
Fig. 7.

Root hair development in grapevine rootstock 140 Ruggeri plants 7 d after treatments were imposed. Root regions with root hairs either (A) exhibiting or (B) not ferric chelate reduction activity were marked and examined under a microscope for root hair development regarding their density and length. Note the subapical to root tip localization of ferric chelate reduction activity in A.

Citation: HortScience horts 43, 3; 10.21273/HORTSCI.43.3.685

Root hair development was examined in roots of Fe-deficient and Fe-sufficient plants treated with either 0 or 20 μm Fe 7 d after the initiation of the treatments. Regions of roots, embedded with the agar technique, exhibiting FCR activity or not were marked and examined microscopically for root hair development. Root hair density and length in root regions exhibiting or not of FCR activity did not differ (Fig. 7). Therefore, root hair development appeared to be independent of the existence of reducing capacity. Similar results were obtained in all treatments (data not shown). Furthermore, FCR activity was also found in totally hairless roots, which are a significant portion of the root system.

Determination of leaf FCR activity was achieved with the use of mesophyll protoplasts, which were isolated from the upper fully expanded leaves of Fe-deficient and Fe-sufficient rootstock plants growing in vitro. Iron deficiency did not alter protoplast intactness (Table 2). Ferric reduction activity in protoplasts was determined immediately after isolation using an assay medium with the appropriate osmoticum to maintain protoplasts intact and physiologically active. The FCR activity in mesophyll protoplasts originating from Fe-sufficient plants was higher than that measured in protoplasts from Fe-deficient plants (Table 2). Ferric chelate reduction activity in mesophyll protoplasts from Fe-deficient plants was decreased by 43.5% compared with that in protoplasts from Fe-sufficient plant leaves when calculated on a protoplast number basis. On a protein basis, the decrease was 40.3%. Chlorophyll content was significantly decreased in protoplasts from Fe-deficient plants compared with that from Fe-sufficient plants (Table 2). The ratio of total chlorophyll measured in protoplasts from Fe-sufficient and Fe-deficient plants was 1.17, whereas the ratio in the leaves from these plants was 1.15 (data not shown). Consequently, a decrease in chlorophyll content associated with Fe deficiency was similar when calculated on a leaf basis and on a protoplast basis.

Table 2.

Ferric chelate reduction activity and chlorophyll concentration in protoplasts isolated from iron (Fe)-deficient and Fe-sufficient grapevine rootstock 140 Ruggeri leaves.z

Table 2.

Discussion

Grapevine rootstocks show diverse behavior regarding their efficiency to acquire Fe at impaired Fe nutrition conditions (Galet, 1979). In this work, grapevine rootstock 140 Ruggeri was chosen, among others, as a model because of its high tolerance to Fe deficiency stress conditions (Tagliavini and Rombolá, 2001). Experimentation on young micropropagated plants comprises a series of advantages. Homogeneous plant population, rapid and visible plant responses to Fe removal, or addition and plant material free of excess accumulation of Fe in plant tissues when compared with seeds and woody cuttings are some of them. Thus far, most of the experiments in grapevine have been conducted with plants originating from woody cuttings, a plant material that is not homogeneous, because the physiological status of the mother plants differs; therefore, it is possible that variations of Fe levels in the cuttings may seriously affect the experimental results.

To circumvent the limitations of these approaches and obtain more clear evidence concerning root FCR activity in response to the external Fe addition in grapevine rootstock plants with different Fe status, an approach using two distinct homogenous plant populations, Fe-deficient and Fe-sufficient plants, was used. To our knowledge, there is no precedent of root FCR activity determination in Fe-efficient grapevine rootstocks in time course experiments. In a previous study, root FCR activity was measured during time in the efficient vinifera cultivar ‘Cabernet Sauvignon’ and the susceptible to Fe deficiency rootstock Vitis riparia (Jiménez et al., 2007). Iron-deficient plants increased their root FCR values five times gradually within 3 d after Fe supply and then retained activity at high levels with slight fluctuations. On the contrary, Fe-deficient plants not supplied with Fe displayed a low stable state. It has been previously reported that Fe sufficiency caused decreased root FCR activity in the grapevine rootstocks V. riparia and 41B and the vinifera cultivar ‘Cabernet Sauvignon’ (Jiménez et al., 2007; Nikolic et al., 2000). In these studies, Fe was used at a concentration of 90 or 100 μm. It is possible that excessive Fe alters the plant response regarding root FCR activity. Similar results as those reported in the present study have been also obtained with other species such as peach (Gogorcena et al., 2000; 2004), olive (De la Guardia and Alcántara, 2002), Annona (Ojeda et al., 2003), and tomato (Zouari et al., 2001), in which supply of Fe(III) to Fe-deficient plants caused an increase of root ferric reduction activity.

Root FCR activity values were generally determined to be in a range from 100 to 500 nmol Fe(II) g−1 fresh weight/h−1 in grapevine rootstock 140 Ruggeri plants. These values are in accordance with those previously reported in grapevine rootstocks 140 Ruggeri (Bavaresco et al., 1991) and 41B (Nikolic et al., 2000) which is also highly tolerant to Fe deficiency. The same lower values of root FCR have been also reported for other perennial plant species (Bohórquez et al., 2001; De la Guardia and Alcántara, 2002; Gogorcena et al., 2000; Ojeda et al., 2003).

It appears that Fe is required for root FCR activity induction in grapevine, which is not a transient effect because Fe-sufficient plants retain their capacity for Fe(III) reduction at high levels even in the presence of Fe. The results also indicate that Fe is required for the induction of FCR activity in mesophyll cells. Two possible explanations are raised as a consequence of the results. The first is that Fe is required as a constituent of the reducing enzymes, because ferric iron reductase is a flavocytochrome (Robinson et al., 1999). The second is that Fe itself acts as a message for the activation of reducing mechanisms when Fe supply is inadequate for plant growth (Schmidt, 2003).

Increased root hair density is a common plant response to Fe deficiency (Schmidt, 1999). Proliferation of root hairs is observed in the areas where reduction activity is located, although not all plants show such a correlation. The results of this study clearly demonstrate that regarding grapevine rootstock 140 Ruggeri, under the described experimental conditions, root hair formation is independent of the Fe status of the plants and FCR activity levels in roots. In a previous report, it has been proposed that Fe reduction is closely related to root hair occurrence within the grapevine rootstocks 140 Ruggeri, SO4, and 101-14 (Bavaresco et al., 1991). However, these findings have not been correlated with the Fe status of the examined rootstock plants. Root hair density and length in Fe-deficient and Fe-sufficient 140 Ruggeri plants did not differ. Levels of FCR activity were also unaffected by the presence of root hairs. It appears that root hair occurrence does not contribute to adaptation mechanisms in the grapevine rootstock 140 Ruggeri under the experimental conditions used in this work that is tolerant to Fe deficiency.

Localization of reduction activity appears to be species-dependent. A number of plants exhibit reduction activity only in the subapical region of the root, whereas in other plants, activity is located throughout the largest part of the roots (Grusak et al., 1999). Grapevine rootstock 140 Ruggeri plants showed a significant shift in the localization pattern of root FCR activity when exposed to Fe(III) in the nutrient solution. The excised roots technique gave equivalent results to the ones observed with the agar-embedded root technique, whereas color intensity matched the measured values. All experiments were conducted with young, white-colored, and nonlignified roots. Nevertheless, what causes the alteration in the distribution patterns of FCR activity in the roots of the grapevine rootstock 140 Ruggeri plants remains vague. It is possible that the level of external Fe supply leads to the expression of different reductase enzyme and electron transport systems. Apart from ferric chelate reductase, other Fe(III)-reducing agents such as exudated reductants (e.g., phenolics and organic acids) may also contribute to the phenomenon (Marschner and Römheld, 1994; Schmidt, 1999).

Measurements of FCR activity on excised root segments, which demonstrate reducing capacity, resulted in magnified values when calculated on a weight basis and compared with total root FCR activity, because parts of the plants’ root system not exhibiting activity were omitted from the assay. The use of excised root apical segments (Brancadoro et al., 1995) may lead to an underestimation of total root FCR activity of Fe-sufficient grapevine plants, because other regions of the lateral roots that also express FCR activity are eventually excluded from the assays. In Fe-sufficient plants, FCR activity, which was measured in the apical regions of the roots, was significantly lower than that measured in the subapical regions. Moreover, apical root regions of Fe-deficient plants presented higher values of FCR activity than the apical regions in Fe-sufficient plants. The results imply that the excised root apical segments technique should be used with caution, because root heterogeneity and diverse FCR localization patterns could create artifacts during measurements of root FCR activity in grapevine.

Mesophyll protoplasts provide a consistent and convenient tool for leaf FCR activity determination, because the total plasma membrane surface is exposed to the assay medium, a condition that cannot be accomplished with other techniques. Measurement of reducing capacity in intact leaves raises experimental difficulties in monitoring Fe(III) reduction activity of intact leaf mesophyll, therefore confining the measurements to the leaf xylem (Kosegarten et al., 1999). The use of leaf disks in grapevine is also limited as a result of difficulties in peeling the abaxial epidermis. In this case, vacuum infiltration is needed, which, however, may cause artifacts as a result of broken cells, modifications of the mesophyll tissue metabolism (Larbi et al., 2001), and uncertainty with regard to the exposure of equal number of cells to the assay medium. It has been suggested that Fe(III) reduction in sugar beet mesophyll protoplasts is light-dependent (González-Vallejo et al., 2000). The results in this article clearly demonstrate that Fe deficiency caused a significant decrease of leaf FCR activity in grapevine rootstock 140 Ruggeri despite the fact that protoplast isolation and assays were conducted in the dark. Further research is needed to prove whether light affects ferric reduction activity in grapevine mesophyll protoplasts.

Iron deficiency decreased leaf chlorophyll index and chlorophyll content in mesophyll protoplasts. Leaf chlorosis appears to be associated with the low root and leaf FCR activity found in Fe-deficient rootstock plants. It has been suggested that chlorophyll content is well correlated with Fe(II) availability in leaves of grapevine (Chen et al., 2004; Römheld, 2000); therefore, it can be used as an indicator of the available Fe status of the leaf cells in grapevine species and cultivars.

In conclusion, Fe(III) affects positively the root and leaf FCR activity in the grapevine rootstock 140 Ruggeri, which is tolerant to Fe deficiency, causing the plants to maintain a stable elevated capacity for Fe(III) reduction. On the contrary, reduction activities under Fe deficiency conditions decline gradually to low levels. Mesophyll protoplasts present a convenient tool for leaf FCR activity determination. The results clearly indicate that the absolute absence of Fe diminishes FCR activity of rootstock 140 Ruggeri both in roots and leaves. Nevertheless, Fe is present at various forms and concentrations in soils even under conditions that do not favor Fe availability, like in calcareous soils, in the presence of HCO3 and at high pH values. Research should extend to studies on the expression of adaptive mechanisms in grapevine under the previously mentioned conditions.

Literature Cited

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    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Fraschini, P. & Perino, A. 1993 Effect of the rootstock on the occurrence of lime-induced chlorosis of potted Vitis vinifera L. cv. ‘Pinot blanc’ Plant Soil 157 305 311

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Fregoni, M. & Fraschini, P. 1991 Investigations on iron uptake and reduction by excised roots of different grapevine rootstocks and a V. vinifera cultivar Plant Soil 130 109 113

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Fregoni, M. & Perino, A. 1994 Physiological aspects of lime-induced chlorosis in some Vitis species. I. Pot trial on calcareous soil Vitis 33 123 126

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Giachino, E. & Pezzutto, S. 2003 Grapevine rootstock effects on lime-induced chlorosis, nutrient uptake, and source-sink relationships J. Plant Nutr. 26 1451 1465

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L. & Poni, S. 2003 Effect of calcareous soil on photosynthesis rate, mineral nutrition, and source-sink ratio of table grape J. Plant Nutr. 26 2123 2135

    • Search Google Scholar
    • Export Citation
  • Bohórquez, J.M., Romera, F.J. & Alcántara, E. 2001 Effect of Fe3+, Zn2+ and Mn2+ on ferric reducing capacity and regreening process of the peach rootstock Nemaguard [Prunus persica (L.) Batsch] Plant Soil 237 157 163

    • Search Google Scholar
    • Export Citation
  • Brancadoro, L., Rabotti, G., Scienza, A. & Zocchi, G. 1995 Mechanisms of Fe-efficiency in roots of Vitis spp. in response to iron deficiency stress Plant Soil 171 229 234

    • Search Google Scholar
    • Export Citation
  • Brüggemann, W., Maas-Kantel, K. & Moog, P.R. 1993 Iron uptake by leaf mesophyll cells: The role of the plasma membrane-bound ferric-chelate reductase Planta 190 151 155

    • Search Google Scholar
    • Export Citation
  • Chen, L.-S., Smith, B.R. & Cheng, L. 2004 CO2 assimilation, photosynthetic enzymes, and carbohydrates of ‘Concord’ grape leaves in response to iron supply J. Amer. Soc. Hort. Sci. 129 738 744

    • Search Google Scholar
    • Export Citation
  • De la Guardia, M.D. & Alcántara, E. 2002 A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince, pear and olive genotypes under iron deficiency stress Plant Soil 241 49 56

    • Search Google Scholar
    • Export Citation
  • Galet, P. 1979 A practical ampelography. Grapevine identification Cornell University Press Ithaca, NY

    • Export Citation
  • Gogorcena, Y., Abadía, J. & Abadía, A. 2000 Induction of in vivo root ferric chelate reductase activity in fruit tree rootstock J. Plant Nutr. 23 9 21

    • Search Google Scholar
    • Export Citation
  • Gogorcena, Y., Abadía, J. & Abadía, A. 2004 A new technique for screening iron-efficient genotypes in peach rootstocks: Elicitation of root ferric chelate reductase by manipulation of external iron concentrations J. Plant Nutr. 27 1701 1715

    • Search Google Scholar
    • Export Citation
  • González-Vallejo, E.B., González-Reyes, J.A., Abadía, A., López-Millán, A.F., Yunta, F., Lucena, J.J. & Abadía, J. 1999 Reduction of ferric chelates by leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet Aust. J. Plant Physiol. 26 601 611

    • Search Google Scholar
    • Export Citation
  • González-Vallejo, E.B., Morales, F., Cistué, L., Abadía, A. & Abadía, J. 2000 Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts Plant Physiol. 122 337 344

    • Search Google Scholar
    • Export Citation
  • Grusak, M.A., Pearson, J.N. & Marentes, E. 1999 The physiology of micronutrient homeostasis in field crops Field Crops Res. 60 41 56

  • Hell, R. & Stephan, U.W. 2003 Iron uptake, trafficking and homeostasis in plants Planta 216 541 551

  • Imsande, J. 1998 Iron, sulfur, and chlorophyll deficiencies: A need for an integrative approach in plant physiology Physiol. Plant. 103 139 144

    • Search Google Scholar
    • Export Citation
  • Jiménez, S., Gogorcena, Y., Hévin, C., Rombolá, A.D. & Ollat, N. 2007 Nitrogen nutrition influences some biochemical responses to iron deficiency in tolerant and sensitive genotypes of Vitis Plant Soil 290 343 355

    • Search Google Scholar
    • Export Citation
  • Katsirdakis, K.C. & Roubelakis-Angelakis, K.A. 1992 Modified culture conditions for increased viability and cell wall synthesis in grapevine (Vitis vinifera L. cv. Sultanina) leaf protoplasts Plant Cell Tissue Organ Cult. 28 255 260

    • Search Google Scholar
    • Export Citation
  • Kosegarten, H.U., Hoffmann, B. & Mengel, K. 1999 Apoplastic pH and Fe3+ reduction in intact sunflower leaves Plant Physiol. 121 1069 1079

  • Larbi, A., Morales, F., López-Millán, A.F., Gogorcena, Y., Abadía, A., Moog, P.R. & Abadía, J. 2001 Technical advance: Reduction of Fe(III)-chelates by mesophyll leaf discs of sugar beet. Multi-component origin and effects of Fe deficiency Plant Cell Physiol. 42 94 105

    • Search Google Scholar
    • Export Citation
  • Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. 1951 Protein measurement with the Folin phenol reagent J. Biol. Chem. 193 265 275

  • Lucena, J.J. 2000 Effects of bicarbonate, nitrate, and other environmental factors on iron deficiency chlorosis. A review J. Plant Nutr. 23 1591 1606

    • Search Google Scholar
    • Export Citation
  • Marschner, H. 1995 Mineral nutrition of higher plants 2nd ed Academic Press London, UK

  • Marschner, H. & Römheld, V. 1994 Strategies of plants for acquisition of iron Plant Soil 165 261 274

  • Mengel, K. 1995 Iron availability in plant tissues- iron chlorosis on calcareous soils 389 397 Abadía J Iron nutrition in soils and plants Kluwer Academic Publishers Dordrecht, The Netherlands

    • Search Google Scholar
    • Export Citation
  • Nikolic, M. & Römheld, V. 1999 Mechanism of Fe uptake by the leaf symplast: Is Fe inactivation in leaf a cause of Fe deficiency chlorosis? Plant Soil 215 229 237

    • Search Google Scholar
    • Export Citation
  • Nikolic, M., Römheld, V. & Merkt, N. 2000 Effect of bicarbonate on uptake and translocation of 59Fe in two grapevine rootstocks differing in their resistance to Fe deficiency chlorosis Vitis 39 145 149

    • Search Google Scholar
    • Export Citation
  • Ojeda, M., Schaffer, B. & Davies, F.S. 2003 Ferric chelate reductase activity in roots of two Annona species as affected by iron nutrition HortScience 38 1104 1107

    • Search Google Scholar
    • Export Citation
  • Ojeda, M., Schaffer, B. & Davies, F.S. 2004 Root and leaf ferric chelate reductase activity in pond apple and soursop J. Plant Nutr. 27 1381 1393

  • Robinson, N.J., Procter, C.M., Connolly, E.L. & Guerinot, M.L. 1999 A ferric-chelate reductase for iron uptake from soils Nature 397 694 697

  • Römheld, V. 2000 The chlorosis paradox: Fe inactivation as a secondary event in chlorotic leaves of grapevine J. Plant Nutr. 23 1629 1643

  • Roubelakis-Angelakis, K.A. & Zivanovitc, S.B. 1991 A new culture medium for in vitro rhizogenesis of grapevine (Vitis spp.) genotypes HortScience 26 1551 1553

    • Search Google Scholar
    • Export Citation
  • Schmidt, W. 1999 Mechanisms and regulation of reduction-based iron uptake in plants New Phytol. 141 1 26

  • Schmidt, W. 2003 Iron homeostasis in plants: Sensing and signaling pathways J. Plant Nutr. 26 2211 2230

  • Schmidt, W., Tittel, J. & Schikora, A. 2000 Role of hormones in the induction of iron deficiency responses in Arabidopsis roots Plant Physiol. 122 1109 1118

    • Search Google Scholar
    • Export Citation
  • Siminis, C.I., Kanellis, A.K. & Roubelakis-Angelakis, K.A. 1994 Catalase is differentially expressed in dividing and nondividing protoplasts Plant Physiol. 105 1375 1383

    • Search Google Scholar
    • Export Citation
  • Smith, B.R. & Cheng, L. 2007 Iron assimilation and carbon metabolism in ‘Concord’ grapevines grown at different pHs J. Amer. Soc. Hort. Sci. 132 473 483

    • Search Google Scholar
    • Export Citation
  • Tagliavini, M. & Rombolá, A.D. 2001 Iron deficiency and chlorosis in orchard and vineyard ecosystems Eur. J. Agron. 15 71 92

  • Vizzotto, G., Matosevic, I., Pinton, R., Varanini, Z. & Costa, G. 1997 Iron deficiency responses in roots of kiwi J. Plant Nutr. 20 327 334

  • Wintermans, J.F.G.M. & De Mots, A. 1965 Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol Biochim. Biophys. Acta 109 448 453

    • Search Google Scholar
    • Export Citation
  • Zouari, M., Abadía, A. & Abadía, J. 2001 Iron is required for the induction of root ferric chelate reductase activity in iron-deficient tomato J. Plant Nutr. 24 383 396

    • Search Google Scholar
    • Export Citation

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Contributor Notes

We thank Dr. George Karabourniotis and Dr. Anastasia Tampakaki for their helpful reviews and constructive comments on the manuscript preparation. We also thank Dr. Constantinos Fasseas for the use of the light microscope and Ioannis Psarokostopoulos for technical assistance with the microscope.

To whom reprint requests should be addressed; e-mail siminis@aua.gr

  • View in gallery

    Ferric chelate reduction activity of intact roots in iron (Fe)-deficient (○) and Fe-sufficient (•) grapevine rootstock 140 Ruggeri plants. Rootstock plants were cultured in nutrient solution with either 0 or 40 μm Fe for 10 d. Assays were run for 2 h and reduction activity was determined at 30-min intervals. Data are means ± se of three different experiments with n = 8. Lines represent linear regression analysis of the means and are different for P ≤ 0.0001 (Fe-deficient, y = –2.5 + 1.557x, slope = 1.557 ± 0.061, r 2 = 0.99; Fe-sufficient, y = 2 + 4.773x, slope = 4.773 ± 0.373, r 2 = 0.96).

  • View in gallery

    Changes in root ferric chelate reduction activity of iron (Fe)-deficient grapevine rootstock 140 Ruggeri plants after Fe supply. Reduction activity was determined in intact roots of plants in nutrient solution supplied either with 0 (○) or 20 (•) μm Fe. Measurements were conducted daily throughout 1 week after treatments were imposed. Data are means ±se of three different experiments with n = 8.

  • View in gallery

    Changes in root ferric chelate reduction activity of iron (Fe)-sufficient grapevine rootstock 140 Ruggeri plants after Fe removal. Reduction activity was determined in intact roots of plants in nutrient solution supplied either with 0 (○) or 20 (•) μm Fe. Measurements were conducted daily throughout 1 week after treatments were imposed. Data are means ±se of three different experiments with n = 8.

  • View in gallery

    Root ferric chelate reduction activity of grapevine rootstock 140 Ruggeri plants growing in nutrient solution with either 0 or 20 μm iron (Fe). Reduction activity was determined in intact roots 14 and 21 d after treatments were imposed to Fe-deficient and Fe-sufficient plants. Data are means ±se of three different experiments with n = 8. Means were separated with t test and * and ** denote significant differences at P ≤ 0.05 and P ≤ 0.01, respectively.

  • View in gallery

    Localization of ferric chelate reduction activity along the lateral roots of grapevine rootstock 140 Ruggeri plants. Experiments were performed in excised roots from plants, treated with either 0 or 20 μm iron (Fe), 7 d after plants were subjected to treatments. Visualization of reduction activity was recorded by embedding the roots in agar medium supplemented with Fe(III) EDTA and ferrozine. Color intensity indicates the degree of reduction activity. The experiment was performed four times with similar results. Bar = 10 mm.

  • View in gallery

    Ferric chelate reduction activity along the lateral roots of grapevine rootstock 140 Ruggeri plants. Data were obtained with excised roots 7 d after iron (Fe) treatments, 0 or 20 μm Fe, were imposed to Fe-deficient plants. The apical (0 to –1.5 cm) and subapical segment (1.5 to 3 cm), both 1.5 cm long, were excised and reduction activity was determined separately. Data are means ±se of two experiments with four replications each. Means were separated with t test and ** and *** denote significant differences at P ≤ 0.01 and P ≤ 0.001, respectively.

  • View in gallery

    Root hair development in grapevine rootstock 140 Ruggeri plants 7 d after treatments were imposed. Root regions with root hairs either (A) exhibiting or (B) not ferric chelate reduction activity were marked and examined under a microscope for root hair development regarding their density and length. Note the subapical to root tip localization of ferric chelate reduction activity in A.

  • Bavaresco, L., Bonini, P. & Giachino, E. 2000 Resistance and susceptibility of some grapevine varieties to lime-induced chlorosis Acta Hort. 528 535 541

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Fraschini, P. & Perino, A. 1993 Effect of the rootstock on the occurrence of lime-induced chlorosis of potted Vitis vinifera L. cv. ‘Pinot blanc’ Plant Soil 157 305 311

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Fregoni, M. & Fraschini, P. 1991 Investigations on iron uptake and reduction by excised roots of different grapevine rootstocks and a V. vinifera cultivar Plant Soil 130 109 113

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Fregoni, M. & Perino, A. 1994 Physiological aspects of lime-induced chlorosis in some Vitis species. I. Pot trial on calcareous soil Vitis 33 123 126

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L., Giachino, E. & Pezzutto, S. 2003 Grapevine rootstock effects on lime-induced chlorosis, nutrient uptake, and source-sink relationships J. Plant Nutr. 26 1451 1465

    • Search Google Scholar
    • Export Citation
  • Bavaresco, L. & Poni, S. 2003 Effect of calcareous soil on photosynthesis rate, mineral nutrition, and source-sink ratio of table grape J. Plant Nutr. 26 2123 2135

    • Search Google Scholar
    • Export Citation
  • Bohórquez, J.M., Romera, F.J. & Alcántara, E. 2001 Effect of Fe3+, Zn2+ and Mn2+ on ferric reducing capacity and regreening process of the peach rootstock Nemaguard [Prunus persica (L.) Batsch] Plant Soil 237 157 163

    • Search Google Scholar
    • Export Citation
  • Brancadoro, L., Rabotti, G., Scienza, A. & Zocchi, G. 1995 Mechanisms of Fe-efficiency in roots of Vitis spp. in response to iron deficiency stress Plant Soil 171 229 234

    • Search Google Scholar
    • Export Citation
  • Brüggemann, W., Maas-Kantel, K. & Moog, P.R. 1993 Iron uptake by leaf mesophyll cells: The role of the plasma membrane-bound ferric-chelate reductase Planta 190 151 155

    • Search Google Scholar
    • Export Citation
  • Chen, L.-S., Smith, B.R. & Cheng, L. 2004 CO2 assimilation, photosynthetic enzymes, and carbohydrates of ‘Concord’ grape leaves in response to iron supply J. Amer. Soc. Hort. Sci. 129 738 744

    • Search Google Scholar
    • Export Citation
  • De la Guardia, M.D. & Alcántara, E. 2002 A comparison of ferric-chelate reductase and chlorophyll and growth ratios as indices of selection of quince, pear and olive genotypes under iron deficiency stress Plant Soil 241 49 56

    • Search Google Scholar
    • Export Citation
  • Galet, P. 1979 A practical ampelography. Grapevine identification Cornell University Press Ithaca, NY

    • Export Citation
  • Gogorcena, Y., Abadía, J. & Abadía, A. 2000 Induction of in vivo root ferric chelate reductase activity in fruit tree rootstock J. Plant Nutr. 23 9 21

    • Search Google Scholar
    • Export Citation
  • Gogorcena, Y., Abadía, J. & Abadía, A. 2004 A new technique for screening iron-efficient genotypes in peach rootstocks: Elicitation of root ferric chelate reductase by manipulation of external iron concentrations J. Plant Nutr. 27 1701 1715

    • Search Google Scholar
    • Export Citation
  • González-Vallejo, E.B., González-Reyes, J.A., Abadía, A., López-Millán, A.F., Yunta, F., Lucena, J.J. & Abadía, J. 1999 Reduction of ferric chelates by leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet Aust. J. Plant Physiol. 26 601 611

    • Search Google Scholar
    • Export Citation
  • González-Vallejo, E.B., Morales, F., Cistué, L., Abadía, A. & Abadía, J. 2000 Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts Plant Physiol. 122 337 344

    • Search Google Scholar
    • Export Citation
  • Grusak, M.A., Pearson, J.N. & Marentes, E. 1999 The physiology of micronutrient homeostasis in field crops Field Crops Res. 60 41 56

  • Hell, R. & Stephan, U.W. 2003 Iron uptake, trafficking and homeostasis in plants Planta 216 541 551

  • Imsande, J. 1998 Iron, sulfur, and chlorophyll deficiencies: A need for an integrative approach in plant physiology Physiol. Plant. 103 139 144

    • Search Google Scholar
    • Export Citation
  • Jiménez, S., Gogorcena, Y., Hévin, C., Rombolá, A.D. & Ollat, N. 2007 Nitrogen nutrition influences some biochemical responses to iron deficiency in tolerant and sensitive genotypes of Vitis Plant Soil 290 343 355

    • Search Google Scholar
    • Export Citation
  • Katsirdakis, K.C. & Roubelakis-Angelakis, K.A. 1992 Modified culture conditions for increased viability and cell wall synthesis in grapevine (Vitis vinifera L. cv. Sultanina) leaf protoplasts Plant Cell Tissue Organ Cult. 28 255 260

    • Search Google Scholar
    • Export Citation
  • Kosegarten, H.U., Hoffmann, B. & Mengel, K. 1999 Apoplastic pH and Fe3+ reduction in intact sunflower leaves Plant Physiol. 121 1069 1079

  • Larbi, A., Morales, F., López-Millán, A.F., Gogorcena, Y., Abadía, A., Moog, P.R. & Abadía, J. 2001 Technical advance: Reduction of Fe(III)-chelates by mesophyll leaf discs of sugar beet. Multi-component origin and effects of Fe deficiency Plant Cell Physiol. 42 94 105

    • Search Google Scholar
    • Export Citation
  • Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. 1951 Protein measurement with the Folin phenol reagent J. Biol. Chem. 193 265 275

  • Lucena, J.J. 2000 Effects of bicarbonate, nitrate, and other environmental factors on iron deficiency chlorosis. A review J. Plant Nutr. 23 1591 1606

    • Search Google Scholar
    • Export Citation
  • Marschner, H. 1995 Mineral nutrition of higher plants 2nd ed Academic Press London, UK

  • Marschner, H. & Römheld, V. 1994 Strategies of plants for acquisition of iron Plant Soil 165 261 274

  • Mengel, K. 1995 Iron availability in plant tissues- iron chlorosis on calcareous soils 389 397 Abadía J Iron nutrition in soils and plants Kluwer Academic Publishers Dordrecht, The Netherlands

    • Search Google Scholar
    • Export Citation
  • Nikolic, M. & Römheld, V. 1999 Mechanism of Fe uptake by the leaf symplast: Is Fe inactivation in leaf a cause of Fe deficiency chlorosis? Plant Soil 215 229 237

    • Search Google Scholar
    • Export Citation
  • Nikolic, M., Römheld, V. & Merkt, N. 2000 Effect of bicarbonate on uptake and translocation of 59Fe in two grapevine rootstocks differing in their resistance to Fe deficiency chlorosis Vitis 39 145 149

    • Search Google Scholar
    • Export Citation
  • Ojeda, M., Schaffer, B. & Davies, F.S. 2003 Ferric chelate reductase activity in roots of two Annona species as affected by iron nutrition HortScience 38 1104 1107

    • Search Google Scholar
    • Export Citation
  • Ojeda, M., Schaffer, B. & Davies, F.S. 2004 Root and leaf ferric chelate reductase activity in pond apple and soursop J. Plant Nutr. 27 1381 1393

  • Robinson, N.J., Procter, C.M., Connolly, E.L. & Guerinot, M.L. 1999 A ferric-chelate reductase for iron uptake from soils Nature 397 694 697

  • Römheld, V. 2000 The chlorosis paradox: Fe inactivation as a secondary event in chlorotic leaves of grapevine J. Plant Nutr. 23 1629 1643

  • Roubelakis-Angelakis, K.A. & Zivanovitc, S.B. 1991 A new culture medium for in vitro rhizogenesis of grapevine (Vitis spp.) genotypes HortScience 26 1551 1553

    • Search Google Scholar
    • Export Citation
  • Schmidt, W. 1999 Mechanisms and regulation of reduction-based iron uptake in plants New Phytol. 141 1 26

  • Schmidt, W. 2003 Iron homeostasis in plants: Sensing and signaling pathways J. Plant Nutr. 26 2211 2230

  • Schmidt, W., Tittel, J. & Schikora, A. 2000 Role of hormones in the induction of iron deficiency responses in Arabidopsis roots Plant Physiol. 122 1109 1118

    • Search Google Scholar
    • Export Citation
  • Siminis, C.I., Kanellis, A.K. & Roubelakis-Angelakis, K.A. 1994 Catalase is differentially expressed in dividing and nondividing protoplasts Plant Physiol. 105 1375 1383

    • Search Google Scholar
    • Export Citation
  • Smith, B.R. & Cheng, L. 2007 Iron assimilation and carbon metabolism in ‘Concord’ grapevines grown at different pHs J. Amer. Soc. Hort. Sci. 132 473 483

    • Search Google Scholar
    • Export Citation
  • Tagliavini, M. & Rombolá, A.D. 2001 Iron deficiency and chlorosis in orchard and vineyard ecosystems Eur. J. Agron. 15 71 92

  • Vizzotto, G., Matosevic, I., Pinton, R., Varanini, Z. & Costa, G. 1997 Iron deficiency responses in roots of kiwi J. Plant Nutr. 20 327 334

  • Wintermans, J.F.G.M. & De Mots, A. 1965 Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol Biochim. Biophys. Acta 109 448 453

    • Search Google Scholar
    • Export Citation
  • Zouari, M., Abadía, A. & Abadía, J. 2001 Iron is required for the induction of root ferric chelate reductase activity in iron-deficient tomato J. Plant Nutr. 24 383 396

    • Search Google Scholar
    • Export Citation
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