SciELO - Scientific Electronic Library Online

vol.31 número1Comparación de tres modelos para estimar la transpiración de un cultivo de jitomate en invernaderoCalidad nutrimental de cuatro abonos orgánicos producidos a partir de residuos vegetales y pecuarios índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados

  • No hay artículos similaresSimilares en SciELO


Terra Latinoamericana

versión On-line ISSN 2395-8030versión impresa ISSN 0187-5779

Terra Latinoam vol.31 no.1 Chapingo ene./mar. 2013



Adaptation strategies of mexican lemon rootstock in response to iron deficiency

Estrategias de adaptación de portainjertos de limón mexicano en respuesta a la deficiencia de hierro

Ranferi Maldonado-Torres1  * 

María Edna Alvarez-Sánchez1 

Jorge D. Etchevers-B.2 

1Departamento de Suelos, Universidad Autónoma Chapingo. 56230 Chapingo, Estado de México, México.

2Colegio de Postgraduados, Campus Montecillo. 56230 Montecillo, Estado de México, México.


In Mexico, alkaline or calcimorphic soils cause iron chlorosis in citrus species, affecting quality and production. Iron chlorosis control through the application of amendments to soil or plants is expensive and has had little success. The use of rootstock adapted to Fe stress conditions is a viable control alternative. In this study, the above mentioned strategy for developing tolerant Mexican lemon was assayed. Volkameriana (Citrus volkameriana Pasq), Macrophylla (Citrus macrophylla Wester) and Sour Orange (Citrus aurantium L.) rootstock were assessed under conditions of restricted Fe to select the varieties with better possibility of adaptation. Plants of each rootstock were grown for 2 months in nutritive solutions adjusted to pH 6.5 or 8 and two levels of Fe,0 and 35μM of Fe. Based on the quantity of absorbed anions and cations, active Fe concentration, OH- or H+ excretion, Fe reductive capacity of the roots and increment in the fresh weight, it was possible to conclude that Volkameriana and Macrophylla rootstocks had better adaptation strategies for acquiring Fe under conditions of deficiency.

Key words: Fe stress; radical excretion; alkaline soil


En México, los cítricos que se desarrollan en suelos alcalinos o calcáreos presentan clorosis férrica disminuyendo el rendimiento y la calidad de la producción. La clorosis férrica ha sido controlada a través de aplicaciones, a la planta o al suelo, de productos que contienen hierro, procedimiento que resulta costoso y poco efectivo. El uso de portainjertos adaptados a condiciones de estrés de Fe es una alternativa viable de control. En esta investigación se evaluó estrategias que desarrollan los portainjertos de limón mexicano, Volkameriana (Citrus volkameriana Pasq), Macrophylla (Citrus macrophylla Wester) y Naranjo agrio (Citrus aurantium L.) para obtener el máximo aprovechamiento de Fe en condiciones restrictivas y seleccionar las variedades con una mayor posibilidad de adaptación. Las plantas de cada patrón se desarrollaron durante 2 meses en soluciones nutritivas ajustando el pH a 6.5 u 8 con dos niveles de Fe, 0 y 35 mM. Con base en la concentración de aniones y cationes absorbidos, el Fe activo, la cantidad de OH- or H+ excretados, la capacidad reductiva de Fe por las raíces y el incremento en el peso fresco, fue posible concluir que los portainjertos Volkameriana y Macrophylla presentaron mejores estrategias de adaptación para la adquisición de Fe en condiciones de deficiencia.

Palabras clave: estrés Fe; excreción radical; suelo alcalino


In Mexico, citrus species develop iron chlorosis when grown in alkaline and calcimorphic soils. In these soils, the availability of iron depends on the kind of iron minerals, particle size, crystalline structure, potential redox, presence of chelating agents and Fe antagonist elements (Cu, Ni and Mn) (Lindsay, 1984; Loeppert, 1986; Srivastava, 2012). Other factors are pH (pH 7.4-8.5), presence of bicarbonates (>200 mg kg-1, high carbonate content (>20%), active CaCO3, clay textures, and absence of oxygen in the radical zone (Chen and Barak, 1982). In conditions of low amounts of available Fe, it has been observed that some plants can develop adaptation strategies to increase their ability to translocate and absorb Fe in their leaves, up to nine times more than others in the same conditions or even in non-deficient conditions (Young and Terry, 1982). Adaptation strategies of plants to Fe deficient conditions may be transitory phenomena, activated before undergoing severe chlorosis when Fe is insufficient (Landsberg, 1984; Brown and Jolley, 1986). Such strategies occur in the subapical root zones, which promote excretion of protons, formation of organic acids (citrate and malate), release of flavine and phenol, increase in reductive capacity and production of ethylene (Römheld and Marschner, 1986; Romera and Alcántara, 1994). Moreover, El-Jendoubi et al. (2012) pointed out that under Fe deficiency, some fruit plants make Fe soluble in the rhizosphere through the excretion of great quantities of malate. Solubilized Fe3+ in the rhizosphere is conducted to the plasma membrane of rhizodermal cells, where it is reduced 10 to 20 times as compared with the situation of non-chlorotic plant (Moog and Brüggemann, 1994; Sueyoshi et al., 1997). Iron (Fe3+) reduction is attributed to a standard reductase constituent of all of the membranes of higher plants and to induced reductase (Bienfait, 1988).

According to Holden et al. (1991), Fe absorption implies that Fe is first reduced in the cell membrane by Fe3+ reductase; after that it goes to the cytoplasm as Fe2+ and is oxidized again forming the complex Fe3+-dicitrate, which travels through the xylem to the aerial part (Chaney, 1989).

After traveling through the xylem and having been previously reduced from Fe3+ to Fe3+ in the membrane, the Fe3+-dicitrate passes to the phloem, and in the cytoplasm of this tissue, it is complexed by nicotinamide for its cytoplasmatic distribution to the required sites (Stephan and Scholz, 1993). Lack of Fe in the foliage decreases the thylakoid stability, chlorophyll synthesis, quantity of carotenes and xantophylls, and photosystem activity, creating a significant reduction in photosynthesis (Römheld and Marschner, 1991) and the presence of visual symptoms of iron chlorosis, which maintains the venation (xylem) and the chlorotic mesophyll green (Abadía et al., 1991). This difference may alter concentrations and nutritive ratios in the foliage; for example, in peach trees (Prunus persica) it was found that the concentration of N, K, Ca, Mg, Mn and K/Ca ratio were higher in Fe-deficient chlorotic leaves than in non-deficient green leaves (Heras et al., 1976). Abadía et al. (1985) observed that the active Fe level of Fe2+, K, and K/Ca and P/Fe ratios were higher in chlorotic leaves of the same species, while in a similar experiment, Köseoglu (1995) registered increments in N, P, K, Mg, increments in P/Fe ratio, and a decrement in total Ca, Zn, Mn, and Fe contents.

For the reasons mentioned above, the objective of this research was to evaluate the adaptive strategies of Mexican lemon rootstocks through observation of changes in nutrients (anions and cations), chlorophyll concentration, active Fe in foliage, OH- or H+ excretion, reductive capacity of the root, and increment of fresh weight rootstocks. This is accomplished through chemical analysis of the plant to evaluate the strategies of response to Fe deficiency. This evaluation is the basis for selecting rootstocks adapted to alkaline soils.

Methods and materials

Fifty seeds of each rootstock species (Citrus macrophylla, Citrus volkameriana and Citrus aurantium) were germinated on Canadian peat in 200 cone polyurethane trays. The seedlings grew with natural light for a month until they reached a height of 10 cm, when they were transplanted to a closed hydroponic system with three liters of nutritive solution containing: 1.3 mM Ca(NO3)2, 1 mM KNO3, 0.8 mM MgSO4, 0.1 mM K2 HPO4, 0.56 μM ZnSO4, 6.7 μM MnSO4, 0.24 μM CuSO4, 33 μM H3BO4 (Manthey et. al.,1994). The treatments were the nutritive solution mentioned above at two levels of Fe, 0 and 35 μM as Fe-EDDHA and two levels of pH, 6.5 and 8. Two plants were set in each recipient and each treatment was replicated five times. Plants were irrigated twice a day, once in the morning and again in the afternoon. The nutritive solution was changed once a week and pH was revised three times per week. In these conditions the plants grew for two months.

At the end of this study, the SPAD units and the concentration of chlorophylls a, b and a+b were determined in young leaves affected by ferric chlorosis. The chlorophylls were determined in 0.5 g of fresh leaves per treatment. Every sample was treated with 40 mL of pure acetone to prepare a homogenized sample that was filtered through fiberglass and the remnant was washed with 40 mL acetone before gauging 100 mL with distilled water. The final extract was composed of an 80% acetone solution, which was refrigerated and protected from the light. Measuring the absorption at 645, 652, and 663 nm, concentration of chlorophylls a and b was determined in the indicated extracts. Chlorophyll absorption was transformed using specific equations proposed by Bruinsma (1963).

Based on the principle that chelate preferably Fe2+ forms a very stable compound, which was determined by a wavelength of 510 nm (Katyal and Sharma, 1980), the active Fe (Fe2+) was extracted with 1-10 ortho-phenanthroline from young leaf samples (Zohlen, 2000).

The reducing capacity was determined in 200 mg of fresh weight of segments of root apex in active growth, through their immersion in 62 mL of a solution of 50 mM of MES buffer (pH 6.3), 1 mM of Ca (NO3)2, 0.8 mM of KNO3, 0.6 mM of MgSO4, 0.3 mM of FeHEDTA and 0.2 mM of bathophenanthroline disulfonic acid (BPDS).The reaction was carried out in darkness for an hour at 33 ºC and in agitation. Reduced Fe was determined through the formation of chelate Fe (BPDS)3 and colorimetrically measured at 536 nm (Manthey et al., 1994).

Nutrient concentration was measured in clean young undamaged leaves. The leaves were placed in plastic bags and transferred to the laboratory in a portable icebox. Each sample was washed according to the procedure proposed by Chapman (1966) and dried at 70 ºC for 48 hours in an oven with forced air circulation before being ground (mesh 40) in a stainless steel mill (Etchevers-Barra, 1988). The material was digested with a bi-acid mixture (HNO3/HClO4 = 4/2 mL) at 203 ºC. In the digested matter, K, Ca, Mg, Fe, Zn, Mn, Cu, and B were determined by spectrophotometry of AES-ICP emission in Varian Liberty II equipment. Nitrogen was digested in acid (H2SO4) and assessed in steam drive distillation (Bremner, 1965) adapted by plant analysis and modified with salicylic acid to include nitrates.

Chlorophyll concentration, active leaf Fe, root reducing capacity, leaf composition, fresh weight (ΔDFW), Σcations and Σanions were plotted and analyzed with ANOVA (Analysis of Variance), and means were compared with the Tukey test (α = 0.05) using the Statistical Analysis System version 6.12 (SAS Institute, 1988) and Microsoft Office ExcelTM.

Results and discussion

Organic and inorganic composition and nutrient balance of cations and anions

The concentration of N-organic was the same in all the rootstocks, while the concentrations of S-organic, NO3-, SO42-, Ca, Mg, Na, ΣC, ΣA, ΣC-ΣA were much higher in Volkameriana than in Macrophylla and Sour Orange. On the other hand, the concentrations of PO43- and K were higher in Macrophylla than in Volkameriana and Sour Orange. Hydroxide ion (OH-) excretion was higher in Sour Orange than in Macrophylla, but Volkameriana excreted H+ instead of OH- (Table 1). These results can be explained by the low levels of Fe in the plants, which promote reactions that modify the plant’s leaf and root structure, morphology, and physiology (CITE). All these strategies are aimed at developing conditions that increase mobilization of Fe in the rhizosphere, increasing absorption and translocation of this element (Brown, 1978) and modifying the concentration of other nutrients in the leaves. The reactions that increased may be H+ excretion, Fe reducing capacity by the roots, formation of radical hair, formation of epidermal “transfer” cells, synthesis and accumulation of organic anions (malate and citrate), flavine excretion and phenol compounds (Bienfait, 1988, 1996).

Table 1 Organic and inorganic composition and nutrimental balance of leaves, developed with or without Fe supplies in solutions with different pH. 

Evaluated variables Rootstocks pH Fe
Volkameriana Macrophyla Sour orange 6.5 8 With Without
--------------------------------------------------------me 100 g-1--------------------------------------------------
N-organic 189.6 a1 177.1 a 185.4 a 185.7 a2 182.4 a 188.0 a3 181.6 a
S-organic 10.3 a 10.0 ab 9.5 b 10.0 a 9.9 a 10.1 a 9.8 a
NO3- 0.7 a 0.4 b 0.3 b 0.4 a 0.5 a 0.4 a 0.5 a
PO43- 3.4 b 4.4 a 3.7 ab 4.2 a 3.5 b 3.9 a 3.8 a
SO42- 7.1 a 4.5 b 4.9 b 5.4 a 5.5 a 5.3 a 5.7 a
K 68.3 b 77.1 a 42.3 c 50.5 b 74.7 a 60.6 b 64.0 a
Ca 104.8 a 83.5 b 65.0 c 71.9 b 97.9 a 87.3 a 81.2 a
Mg 49.5 a 28.5 c 37.6 b 35.2 b 41.9 a 37.9 a 40.3 a
Na 1.7 a 0.4 c 0.4 b 0.4 b 1.2 a 0.7 a 0.9 a
Σ cations(ΣC) 224.4 a 189.6 b 146.5 c 158.2 b 214.8 a 108.1 b 186.6 a
Σ anions (ΣA) 11.4 a 9.4 b 8.9 b 10.2 a 9.6 b 9.7 a 10.1 a
ΣC - ΣA 203.0 a 180.2 b 136.5 c 148.0 b 205.2 a 171.2 b 176.4 a
OH - Excretion -13.0 c 19.6 b 50.0 a 42.1 a -9.9 b 25.3 a 12.5 b

1Horizontal reading, varieties of rootstocks with the sae letter are not greatly different; 2 Horizontal reading, pH levels with different greatly different; 3 Horizontal reading, Fe levels with different letter are greatly different.

The high absorption of cations (K, Ca, Mg, Na), ΣC, ΣC-ΣA (organic anions), observed in Volkameriana and Macrophylla rootstocks, relative to that in Sour Orange, could be determined due to the processes involved during the development of strategies (acidification of the rhizosphere and reductase activity) for adaptation of these rootstocks when there is a scarcity of Fe.

Dakora y Phillips (2002) established that, when Fe is deficient, in Fe-efficient plants radical H+ excretion decreases the absorption of anions and increases both the accumulation of organic anions (ΣC-ΣA) and the absorption of cations. It has also been observed that these plants, when nourished with NO3-, decrease or change radical OH- excretion for H+ excretion but either alcalinization or acidification is present in all the radical system (Marschner and Römheld, 1994).

Most of the K concentration determined in the Volkameriana and Macrophylla rootstocks, relative to Sour Orange, could be attributed to greater excretion of H+ by the root apex, which helps to acidify the rhizosphere, increase the Fe activity around it and exchange it for K+ in order to maintain the electric potential of the membrane (Jolley and Brown, 1985; Römheld and Marschner, 1986). In the leaves affected by ferric chlorosis the synthesis of carbohydrates decreases and as a result the biomass also decreases, causing an increase on potassium level (Hamzé et al., 1986).

The Ca levels determined in Volkameriana were higher with regard to Macrophylla and Sour Orange.

Calcium is an element considered by Cohen et al. (1997) as a regulator of the ferric reductase activities that can favor adaptation of a plant to Fe stress, although there have been results that do not agree, such as those of Köseoglu (1995); the latter author found increments of N, P, K, and Mg, while Ca, Zn, Mn and Fe decreased in Prunus persica leaves affected by ferric chlorosis.

The Mexican lemon rootstocks grew in the solution with pH 6.5 increased the concentration of N-organic, S-organic, PO43-, ΣA and OH excretion; relative to thosegrew in the pH 8.0 solution.

However, K, Ca, Mg, Na concentrations, ΣC and ΣC-ΣA were higher in the rootstocks growing in the solution pH 8. These results coincide with the results reported by Larbi et al. (2010), who pointed out that more cations are absorbed in the interval close to neutral, while more anions are absorbed in the acid interval. The increment in the accumulation of organic anions (ΣC-ΣA) at alkaline pH coincides with the information reported by Smith and Raven (1979), who pointed out that this accumulation is a response of the plant to maintain the cytoplasm pH in the interval 7 to 7.5. Nevertheless, the OH- xcretion was greater at acid pH and there was little H+ production under the alkaline condition, a mechanism of the plant to maintain electro- neutrality to favor Fe activity (Marschner and Römheld, 1994). N-organic, S-organic, NO3-, SO42-, Ca, Mg, Na and ΣA concentrations were not affected by the treatments with or without Fe. However, the K level and the ΣC and the ΣC-ΣA were higher in the treatments without Fe, while the OH- excretion was higher when Fe was applied to the Mexican lemon rootstocks.

The results on Fe partially coincide with the findings of Abadía et al. (1985, 1989) who determined that in peach and pear leaves, affected by a lack of Fe, the levels of P, Ca, Mg, Na, Fe, Mn, Cu and Zn levels did not change significantly with regard to non-chlorotic plants. However, in another study carried out in Prunus persica, by Heras et al. (1976) N, K, Ca, Mg and Mn were concentrated in Fe- deficient chlorotic leaves rather than in non-deficient green leaves. In chlorotic leaves of the same species, the active Fe and K levels increased (Abadía et al., 1985), while in a similar experiment the N, P, K and Mg concentrations increased, and the Ca, Zn, Mn, and Fe concentrations decreased (Köseoglu, 1995). These results clearly show that the nutrient concentrations and sensitivity of leaves to ferric chlorosis may vary among plant species (Figure 1 and 2). The tree rootstocks had an increment in K, Ca, Mg, and Na absorption, when pH was modified from 6.5 to 8. But in the treatments without Fe, Volkameriana consistently showed an increase in the quantity of absorbed cations, except for Ca. Of the four cations, except K, Volkameriana absorbed more Ca, Mg and Na than Macrophylla and Sour Orange (Figure 1 and 2). According to Cohen et al. (1997), the greatest absorption of cations (K, Ca, Mg, Mn and Zn) was associated with the greatest activity of ferric reductases, which, regulated by these, decreased ferric chlorosis.

Figure 1 pH effect, with (+) or without (-) Fe application on the concentration of: (a) K, (b) Ca, (c) Mg and (d) Na, in different rootstocks of Mexican lemon. DW = dry weight. 

Figure 2 pH effect, with (+) or without (-) Fe application on the concentration of: (a) Σ cations, (b) Σ anions, (c) Σ cations- Σ anions and (d) OH- excretion in different rootstocks of Mexican lemon. 

The Σ Cations and the ΣCations-ΣAnions (organic anions) were higher in Volkameriana, followed by Macrophylla and Sour Orange. In Volkameriana and Macrophylla, the increments were even higher when pH changed from 6.5 to 8, and such increments were even higher in the treatments where Fe was suppressed (Figure 2).

Volkameriana presented a similar anion absorption in the three treatments where Fe was shortly available (pH 6.5-Fe, pH 8+Fe, pH 8-Fe). In contrast, Macrophylla showed a decrease in anion absorption when pH changed from 6.5 to 8, while Orange Sour did not show a clear trend. In general, the results were similar to those reported by Egmond and Aktas (1977), who obtained a decrease in anion absorption in Fe-deficient tomato and bean plants.

It was observed that at pH 6.5, Volkameriana and Macrophylla decreased OH- excretion when Fe was suspended, while at pH 8, they excreted H+. On the other hand, Sour Orange only excreted OH- . In all the treatments no trend appeared (Figure 2).

It is important to mention that Volkameriana always showed the lowest levels in OH- production and excretion, with regard to Macrophylla and Sour Orange, and greater H+ production than Macrophylla, as it is shown in Figure 2 d. This phenomenon is important because, according to Römheld et al. (1984) and Römheld and Marschner (1986), H+ excretion allows acidification of the root’s interphase and promotes Fe solubilization, reductase activity, and phenol release. These processes help the plant to better utilize Fe. Egmond and Aktas (1977) have studied OH- and H+ excretion in plants subjected to Fe deficiency and determined that plants that excreted low levels of OH- showed less ferric chlorosis, and they even showed a change in the ionic balance in favor of H+ excretion. Abadía et al. (2002) also says that plants subjected to Fe stress and acidify the rhizosphere show an increased level of organic acids (citrate and malate), which contribute to greater acidification of the rhizosphere when exuded and generate reductive power, controlling the cation/anion balance in order to regulate cell pH and increase Fe transport from the root to the aerial part (Egmond and Aktas, 1977; Landsberg, 1986). Acidification alone may influence the pH of the first 2 mm of the root surface, increasing the reductase activity of the cell membrane, weakening the Fe-O bonds and releasing the metal, even in calcimorphic soils (Schwertmann, 1991; Susín et al., 1996).

Minor elements, nutrient ratios, root reductive capacity and increase of fresh weight in rootstocks with Fe stress

Volkameriana showed a significantly higher level of active Fe than Macrophylla and Sour Orange (Table 2). In Volkameriana, concentrations similar to those reported by Mohammad et al. (1998) in lemon leaves were determined. The authors mentioned above determined concentrations of 11 mg kg-1 of active Fe in chlorotic leaves and concentrations of 17 mg kg-1 in leaves with moderate chlorosis. The results of active Fe found in our research in Macrophylla and Sour Orange were similar in concentration to those reported by Mohammad et al. (1998) for chlorotic leaves. While Volkameriana showed an active Fe concentration similar to that reported by the author mentioned for moderately chlorotic leaves. The reason for determining active Fe, according to Zohlen (2000), is that it correlates better with chlorophyll concentration than with total Fe. Richier et. al. (2012) explains that this is due to the function of Fe2+ associated with chloroplast development, protein formation (cytochromes), ferredoxin, electron transport chain, and chloroplast mRNA and rRNA. The results showed a higher concentration of this element in the treatments with pH 6.5 and with Fe, than those with pH 8 and without Fe. The lower concentration of active Fe at pH 8 may be attributed to a lower quantity of available Fe, according to Lindsay and Schwab (1982), who pointed out that for every unit of increase in pH the dissolved Fe activity decreases thousand times because of the balance between solubility and precipitation. At pH lower than 7.4, ferrihydrite dominates, between 7.4 and 8.5 goethite dominates, while at a pH above 8.5 ferric hydroxides dominate; all of these compounds are of low solubility (Loeppert, 1986). According to Lindsay and Schwab (1982), the lowest concentration of Fe (10-10.4 M) is reached at a pH between 7.4 and 8.5, that is, 250 times lower than the critical level (10-8 M ) in which plants such as lemon may present Fe deficiency. At pH 8, the reductive capacity of the root is low and, consequently, the transfer of Fe to specific sites is also low (Romera et al., 1991; Fox et al., 1996).

Table 2 Microelements concentration, nutrient rations, chlorophyll concentration, Fe reduced by the roots and increment of fresh weight of rootstocks of Mexican lemon developed at different pH and with or without Fe. 

Evaluted variables Rootstocks pH Fe
Volkameriana Macrophyla Sour orange 6.5 8 With Without
Active Fe (mg kg-1) 17.1 a1 10.0 b 11.1 b 14.4 a2 11.1 b 15.7 a3 9.8
Fe (mg kg-1) 23.3 a 26.0 a 22.9 a 25.5 a 23.1 a 24.8a 23.4
Mn (mg kg-1) 33.7 a 36.1 a 14.1 b 29.1 a 27.2 a 24.5 b 31.4
Zn (mg kg-1) 25.8 a 21.8 ab 19.1 b 22.6 a 22.0 a 22.2 a 22.2
Cu (mg kg-1) 15.9 a 15.0 a 14.4 a 15.5 a 14.5 b 15.2 a 15.0
B (mg kg-1) 221.0 a 116.3 b 66.3 c 132.4 a 136.0 a 149.4 a 119.6
K/Ca4 1.7 a 1.2 b 1.3 b 1.4 a 1.5 a 1.5 a 1.4
K/Zn4 1043.6 b 1415.4 a 884.8 b 940.1 b 1288.9 a 1072.9 a 1156.2
P/Fe4 49.7 a 56.0 a 52.0 a 53.4 a 51.9 a 54.7 a 50.7
μmoles Fe2 + h-1 g FW 35.5 ab 36.2 a 25.2 b 36.3 a 28.8 b 28.8 b 35.8
Chlorophyll a (mg L-1) 4.1 a 3.9 a 3.2 a 4.8 a 3.9 a 4.8 a 4.0
Chorophyll b (mg L-1) 7.7 a 5.4 b 4.6 b 6.5 a 5.5 a 6.5a 5.5
Chorophyll a+b (mg L-1) 13.6 a 9.5 b 8.1 b 11.8 a 10.0 a 11.7 a 10.1
∆ Fresh weight (g) 31.0 b 42.5 a 13.0 c 32.3 a 25.8 b 29.3a 28.8

1 Horizontal reading. Rootstock varieties with the same letter are not significantly different; 2 Horizontal reading. pH levels with different letter are significantly different; 3 Horizontal reading. Fe levels with different setter are significantly different; 4 P, K and Ca concentrations in per cent and Fe and Zn in regarding dry weight.

Total Fe concentration in the evaluated rootstocks did not show any significant difference. In different studies, it has been determined that this element does not match chlorophyll concentrations because it is being accumulated in inactive form in chlorotic leaves (Abadía et al., 1989).

Inactive iron accumulation originates total Fe concentrations equal to and higher than the concentrations that have been found in green leaves (Morales et al., 1998), and the plant may recover chlorophyll concentrations by activation with acid solutions sprayed on the foliage (Tagliavini et al., 1997). Neither pH nor Fe application caused significant difference in total Fe concentration. This may be explained by the existence of root factors and strategies developed by plants that determine its availability, absorption, and transfer (Kosegarten et al., 1999).

Volkameriana and Macrophylla had higher concentrations of Mn and Zn than Sour Orange. This may be attributed not only to the effect of acidification of the rhizosphere that both rootstocks promoted, but also to the simultaneous effect of the formation of “transfer” cells in the root apex that favor the absorption of these elements. According to Jolley and Brown (1985), Hydrogen ion excretion and the radical apical formation of “transfer” cells coincide in time and space; this phenomenon demands K, Mn, Zn and Fe absorption to maintain membrane electrical potential.

Moreover, concentration of Cu, P/Fe ratio and chlorophyll did not show significant differences among the rootstocks and Fe supply. However, concentration of Cu was affected by pH. The K/Ca ratio was significantly higher in Volkameriana than in Macrophylla and Sour Orange (Table 2). This ratio has been used in citrus as an indicator of resistance to Fe deficiency. It has been said that a ratio higher than 1.2 is high and is associated with high resistance to ferric chlorosis, while a lower ratio is considered low and indicates ferric chlorosis sensitivity (Hamzé et al., 1986).

Volkameriana showed a high ratio making it the species with the highest resistance to ferric chlorosis, while Macrophylla is borderline and Sour Orange is slightly above the level indicating resistance to ferric chlorosis. pH and Fe dose promoted values of K/Ca ratio above the limits for resistance to ferric chlorosis; however, there was no statistically significant difference from the effect of these factors.

The K/Zn ratio was higher in Macrophylla than in Volkameriana and Sour Orange (Table 2). This ratio has been used as an indicator of Fe-deficiency (Igartua et al., 2000) because the K concentration increases and the Zn levels decrease in chlorotic plants, resulting in higher and more constant ratios than individual values over the years (Belkhodja et al., 1998).

The root’s reductive capacity was significantly higher in Macrophylla and Volkameriana than in Sour Orange. Reductive capacity is attributed to specific reductase located in the membrane of root’s apical cells, which in plants subjected to Fe deficiency increases their activity 10 to 20 times than in non-deficient plants (Sueyoshi et al., 1997). The results obtained in this study coincide with those of Hamzé et al. (1986) and Manthey et al., (1994). Based on the root’s reductive capacity, Volkameriana and Macrophylla are considered resistant to Fe stress, while Sour Orange is classified as sensitive. When considering only the pH effect or the Fe dose, higher values of reductive capacity in treatments at pH 6.5 and without Fe were obtained. According to Susín et al. (1996), the highest root reductive power due to higher reductase activity occurs around pH 5.5, and it increases under Fe deficiency (Sueyoshi et al., 1997).

Regarding chlorophyll concentration, Volkameriana presented significantly higher levels of chlorophylls b and a+b than Macrophylla and Sour Orange (Table 2). This result can be attributed to the high reductive capacity of the radical system in Volkameriana, which allowed Fe2+ absorption and traslocation, important steps in chlorophyll synthesis (Terry and Zayed, 1995). Some studies have found that active Fe regulates chloroplastic protein synthesis, important in the formation of the complex chlorophyll-proteins, which constitutes the thylakoid membrane. For this reason, photosynthetic pigment synthesis (chlorophyll and carotenoids) can be negatively affected by up to 95% because of a decrease in Fe2+ (Fodor et al., 1995; Zholem, 2000). In our study, it was found that Volkameriana had high reductive capacity in roots, higher levels of active Fe in foliage, and coincidently higher levels of chlorophyll a+b. These facts allow classifying this rootstock as that having the best response to Fe deficiency. Regarding the pH effect and the Fe dose, there were no significant differences.

Most of the increments in fresh matter registered in Macrophylla and Volkameriana rootstocks are the result of Fe-deficiency response strategies such as more reductive capacity of the root, reduction in OH- excretion, increase in H+ excretion, more K, Ca, Na, Mn, Zn, and B absorption, high K/Ca, K/Zn and active Fe ratios that originated higher concentrations of chlorophylls b and a+b.

The K/Ca ratio in Volkameriana was very similar in the four treatments; in Macrophylla this ratio increased when pH changed from 6.5 to pH 8, and in Sour Orange this ratio was lower in the treatments without Fe at pH 6.5 and pH 8. Volkameriana rootstock showed a more stable value of K/Ca ratio, regardless of the treatment, while in Macrophylla and Sour Orange there was a greater variation (Figure 3), which, according to Hamzé et al. (1986), is associated with lower resistance to ferric chlorosis.

Figure 3 pH effect, with (+) or without (-) Fe application on the concentration of: (a) K/Ca, (b) K/Zn, (c) Mn and (d) B, in different rootstocks of Mexican lemon. 

The K/Zn ratio tended to be higher when the pH of the nutritive solution was increased, and it was higher in Macrophylla followed by Volkameriana and Sour Orange. Mn absorption was higher when pH increased from 6.5 to 8. It was significantly higher in both Volkameriana and Macrophylla rootstocks (Figure 3) due to the activity of ferric reductases and H+ excretion (Sijmons and Bienfait, 1986; Cohen et al., 1997).

In the three rootstocks, B increased when pH changed from 6.5 to 8 (Figure 3); however, it was much higher in Volkameriana rootstock due to B participation in cell division (Larbi et al., 2010). In Fe-deficient plants, B must be highly required because side root formation (Moog et al., 1995), radical hairs, and apex enlargement are promoted (Landsberg, 1996).

The lowest levels of active Fe were found at pH 6.5 without Fe and at pH 8 with Fe. On the other hand, the highest levels of active Fe were found at pH 6.5 with Fe and at pH8 without Fe, being Volkameriana the rootstock with the highest concentration of active Fe (Figure 4).

Figure 4 Active Fe-, root’s reductive capacity, chlorophyll concentration, and increase in plant’s fresh weight (DFW) to the different treatments of rootstocks of Mexican lemon. 

When Fe supply was eliminated, the root’s reductive capacity at pH 6.5 increased in Volkameriana and Sour Orange rootstock, and an equal decrease at pH 8 was observed in treatments with or without Fe. At pH 6.5 and without Fe the same reductive capacity was observed (Figure 4), which coincides with the information provided by Sueyoshi et al. (1997) and González-Vallejo et al. (1999), who state that the best pH for promoting more activity of the membrane’s ferric reductases in beet leaves was between 6.5 and 7, increasing under Fe- deficient conditions. According to Bienfait (1996), H+ excretion is related to citrate accumulation. Citrate is isomerized in the form of a-oxoglutarate and reduced NADP+ (NADPH) which directly or indirectly transfers electrons to the membrane ferric reductases that are responsible for reducing Fe.

Furthermore, the concentration of chlorophyll a+b showed higher levels at pH 6.5 than pH at 8 with or without Fe; this concentration was higher in Volkameriana in all the treatments. Moreover, a direct ratio (or relationship) between the quantity of active Fe and the roots’ reductive capacity regarding the concentration of chlorophyll a+b was observed. Finally, the ΔFW of fresh weight was higher in Macrophylla and Volkameriana rootstocks than in Sour Orange. Macrophylla showed less growth when the pH increased and Fe was not applied, while Volkameriana had constant values in the four treatments. Sour Orange showed a very irregular increase in fresh weight in the different treatments, but it was always low (Figure 4).


  • The results obtained in this study showed higher leaf concentrations of N-organic, S-organic, NO3 -, SO42-, Ca, Mg, Na, ΣCations, ΣAnions, ΣCations-ΣAnions, active Fe, Zn, Cu, B, higher concentration of chlorophylls a, b and a+b, and K/Ca ratio in Volkameriana, than in Macrophylla and Sour Orange (was this difference a significative one). Macrophylla, however, showed higher levels of PO43+, K, total Fe, Mn, K/Zn ratio, root reductive capacity and increase of fresh weight than Volkameriana and Sour Orange.

  • Volkameriana and Macrophylla decreased OH- excretion in conditions of limited Fe, while extreme Fe- deficiency promoted H+ excretion. For this reason, nutrimental element absorption improved. These rootstocks developed better strategies to respond to Fe stress by promoting changes in leaf nutrient composition and reductive capacity in the root, originating better utilization of Fe to maintain higher levels of chlorophylls.

Literature cited

Abadía, A., A. Poc, and J. Abadía. 1991. Could iron nutrition status be evaluated though photosynthetic pigment change? J. Plant Nutr. 14: 987-999. [ Links ]

Abadía, A., M. Sanz, J. de las Rivas, and J. Abadía. 1989. Photosynthetic pigments and mineral composition of iron deficient pear leaves. J. Plant Nutr. 12: 827-838. [ Links ]

Abadía, J., A. López-Millán, A. Rombolà, and A. Abadía. 2002. Organic acids and Fe deficiency: a review. Plant Soil 241: 75-86. [ Links ]

Abadía, J., J. N. Nishio, E. Monge, L. Montañés, and L. Heras 1985. Mineral composition of peach leaves affected by iron chlorosis. J. Plant Nutr. 8: 697-707. [ Links ]

Belkhodja, R., F. Morales, M. Sanz, A. Abadía, and J. Abadía. 1998. Iron deficiency in peach trees: effects on leaf chlorophyll and nutrient concentration in flowers and leaves. Plant Soil 203: 257-268. [ Links ]

Bienfait, H. F. 1988. Mechanisms in Fe-efficiency reactions of higher plants. J. Plant Nutr. 11: 605-629. [ Links ]

Bienfait, H. F. 1996. Is there a metabolic link between H+ excretion and ferric reduction by roots of Fe-deficient plants? - A viewpoint. J. Plant Nutr. 19: 1211-1222. [ Links ]

Bremner, J. M. 1965. Total nitrogen. pp. 1149-1178. In: C. A. Black (ed.). Methods of soil analysis (Part 2) (Agronomy 9). American Society Agronomy. Madison, WI, USA. [ Links ]

Brown, J. C. 1978. Mechanism of iron uptake by plants. Plant Cell Environ. 1: 249-257. [ Links ]

Brown, J. C. and D. von Jolley. 1986. An evaluation of concepts related to iron-deficiency chlorosis. J. Plant Nutr. 9: 175-186. [ Links ]

Bruinsma, J. 1963. Absorption of light by chlorophyll a and b in plant extracts. Photochem. Photobiol. 2: 241-249. [ Links ]

Chaney, R. L. 1989. Translocation of iron roots to shoot. En 5th International Symposium of Iron Nutrition and Interactions in Plants. Jerusalem, Israel. [ Links ]

Chapman, H. D. 1966. Leaf and soil analysis in citrus orchards. Manual 25. University of California, Div. Agr. Sci., Agric. Experiment Station-Extension Service. Berkeley, CA, USA. [ Links ]

Chen, Y. and P. Barak. 1982. Iron nutrition of plants in calcareous soils. Adv. Agron. 35: 217-238. [ Links ]

Cohen, C. K., W. A. Norvell, and L. V. Kochian. 1997. Induction of the root cell plasma membrane ferric reductase (an exclusive role for Fe and Cu). Plant Physiol. 114: 1061-1069. [ Links ]

Dakora, D. F. and D. A. Phillips. 2002. Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245: 35-47. [ Links ]

Egmond, F. van and M. Aktas. 1977. Iron-nutritional aspects of the ionic balance of plants. Plant Soil 48: 685-703. [ Links ]

El-Jendoubi, H., E. Igartua, J. Abadía, andA. Abadía. 2012. Prognosis of iron chlorosis in pear (Pyrus communis L.) and peach (Prunus persica L. Batsch) trees using bud, flower and leaf mineral concentrations. Plant Soil 354: 121-139. [ Links ]

Etchevers-Barra, J. D. 1988. Manual de métodos de análisis de suelos, plantas, aguas y fertilizantes. Centro de Edafología, Colegio de Postgraduados en Ciencias Agrícolas. Montecillo, Edo. de México. [ Links ]

Fodor, F., B. Böddi, E. Sárvári, G. Záray, E. Cseh, and F. Láng. 1995. Correlation of iron content, spectral forms of chlorophyll and chlorophyll-proteins in iron deficient cucumber (Cucumis sativus). Physiol. Plant 93: 750-756. [ Links ]

Fox, T. C., J. E Shaff, M. A. Grusak, W. A. Norvell, Y. Chen, R. L. Chaney, andL. V. Kochian. 1996. Direct measurement of 59Fe labeled Fe2+ influx in root pea using a chelator buffer system to control free Fe2+ in solution. Plant Physiol. 111: 93-100. [ Links ]

González-Vallejo, E. B., J. A. González-Reyes, A. Abadía, A. F. López-Millán, F. Yunta, J. J. Lucena and J. J. Abadía. 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. [ Links ]

Hamzé, M., J. Ryan, and M. Zaabout. 1986. Screening of citrus rootstocks for lime-induced chlorosis tolerance. J. Plant Nutr. 9: 459-469. [ Links ]

Heras, L., M. Sanz, and L. Montañes 1976. Corrección de la clorosis férrica en melocotonero y su repercusión sobre el contenido mineral, relaciones nutritivas y rendimientos. An. Estac. Exp. Aula Dei 13: 261-289. [ Links ]

Holden, M. J., D. G. Luster, R. L. Chaney, T. J. Buckout, and C. Robinson. 1991. Fe3+-chelate reductase activity of plasma membranes isolated from tomato (Lycopersicon esculentum Mill.) roots. Comparison of enzymes from Fe-deficient and Fe-sufficient roots. Plant Physiol. 97: 537-544. [ Links ]

Igartua, E., R. Grasa, M. Sanz, A. Abadía , and J. Abadía. 2000. Prognosis of iron chlorosis from the mineral composition of flowers in peach. J. Hortic. Sci. Biotechnol. 75: 111-118. [ Links ]

Jiménez, A, A. Torrecillas, F. Sevilla, M. F. Ortuño, W. Conejero, F. Ferreres, S. Medina, A. Galindo, and A. Gil-Izquierdo. 2012. Lime-induced iron chlorosis in citrus: Diagnosis through physiological and metabolic evidences. pp. 321-332. In: A. K. Srivastava. Advances in citrus nutrition. Springer. Dordrecht, The Netherland. [ Links ]

Jolley, V. D. and J. C. Brown 1985. Iron stress response in tomato affected by potassium and renewing nutrient solutions. J. Plant Nutr. 8: 527-541. [ Links ]

Katyal, J. C. and B. D. Sharma. 1980. A new technique of plant analysis to resolve iron chlorosis. Plant Soil 55: 105-119. [ Links ]

Kosegarten, H., B. Hoffmann, and K. Mengel. 1999. Apoplastic pH and Fe3+ reduction in intact sunflower leaves. Plant Physiol. 121: 1069-1079. [ Links ]

Köseoglu, A. T. 1995. Effect of iron chlorosis on mineral composition of peach leaves. J. Plant Nutr. 18: 765-776. [ Links ]

Landsberg, E. C. 1984. Regulation of iron-stress-response by whole- plant activity. J. Plant Nutr. 7: 609-621. [ Links ]

Landsberg, E. C. 1986. Function of rhizodermal transfer cells in the Fe stress response mechanisms of Capsicum annuum L. Plant Physiol. 82: 511-517. [ Links ]

Landsberg, E. C. 1996. Hormonal regulation of iron-stress response in sunflower roots: a morphological and cytological investigation. Protoplasma 194: 69-80. [ Links ]

Lindsay, W. L. 1984. Soil and plant relationships associated with iron deficiency with emphasis on nutrient interactions. J. Plant Nutr. 7: 489-500. [ Links ]

Lindsay, W. L. and A. P. Schwab. 1982. The chemistry of iron in soil and its availability to plants. J. Plant Nutr. 5: 821-840. [ Links ]

Loeppert, R. H. 1986. Reactions of iron and carbonates in calcareous soils. J. Plant Nutr. 9: 195-214. [ Links ]

Manthey, J. A., D. L. McCoy, and D. E. Crowley 1994. Stimulation of rhizosphere iron reduction and uptake in response to iron deficiency in citrus rootstocks. Plant Physiol. Biochem. 32: 211-215. [ Links ]

Marschner, H. and V. Römheld. 1994. Strategies of plants for acquisition of iron. Plant Soil 165: 261-274. [ Links ]

Mohammad, M. J., H. Najim, and S. Khresat. 1998. Nitric acid-and o-phenanthroline-extractable iron for diagnosis of iron chlorosis in citrus lemon trees. Commun. Soil Sci. Plant Anal. 29: 1035-1045. [ Links ]

Moog, P. R. and W. Brüggemann 1994. Iron reductase systems on the plant plasma membrane-A review. Plant Soil 165: 241-260. [ Links ]

Moog, P. R., T. van der Kooij, W. Brüggemann, J. W. Schiefelbein, and P. J. Kuiper. 1995. Responses to iron deficiency in Arabidopsis thaliana: the turbo iron reductase does not depend on the formation of root hairs and transfer cells. Planta 195: 505-513. [ Links ]

Morales, F., R. Grasa, A. Abadía, and J. Abadía 1998. Iron chlorosis paradox in fruit trees. J. Plant Nutrit. 21: 815-825. [ Links ]

Richier, S., A. I. Macey, N. J. Pratt, D. J. Honey, C. M. Moore, and T. S. Bibby. 2012. Abundances of iron-binding photosynthetic and nitrogen-fixing proteins of trichodesmium both in culture and in situ from the North Atlantic. Plos One 7(5):e35571. doi:10.1371/journal.pone.0035571. [ Links ]

Romera, F. J. and E. Alcántara 1994. Iron-deficiency stress responses in cucumber (Cucumis sativus L.) roots (a possible role for ethylene?). Plant Physiol. 105: 1133-1138. [ Links ]

Romera, F. J., E. Alcántara, and M. D. de la Guardia. 1991. Characterization of tolerance to iron chlorosis in different peach rootstocks grown in nutrient solution. I. Effect of bicarbonate and phosphate. Plant Soil 130: 115-119. [ Links ]

Römheld, V. and H. Marschner. 1986. Mobilization of iron in the rhizosphere of different plant species. pp. 155-204. In: P. B. Tinker and A. Läuchli (eds.). Advances in plant nutrition vol. 2. Praeger Scientific. New York, NY, USA. [ Links ]

Römheld, V. and H. Marschner. 1991. Function of micronutrients in plant. pp. 297-328. In: J. J. Mortvedt, F. R. Cox, L. M. Shuman, and R. M. Welch (eds.). Micronutrients in agriculture. Soil Science Society of America. Madison, WI, USA. [ Links ]

Römheld, V., C. Müller, and H. Marschner 1984. Localization and capacity of proton pumps in roots of intact sunflower plants. Plant Physiol. 76: 603-606. [ Links ]

SAS Institute. 1988. SAS-STAT user’s guide. Release 6.03. SAS Institute. Cary, NC, USA. [ Links ]

Schwertmann, U. 1991. Solubility and dissolution of iron oxides. Plant Soil 130: 1-25. [ Links ]

Sijmons, P. C. and H. F. Bienfait. 1986. Development of iron III ion reduction activity and hydrogen ion extrusion during growth of iron-deficient bean (Phaseolus vulgaris cultivar Prelude) plants in a rhizostat. Biochem. Physiol. Pflanz. 181: 283-299. [ Links ]

Smith, F. A. and J. A. Raven 1979. Intracellular pH and its regulation. Ann. Rev. Plant Physiol. 30: 289-311. [ Links ]

Stephan, U. W. and G. Scholz 1993. Nicotianamine: mediator of transport of iron and heavy metals in the phloem? Plant Physiol. 88: 522-529. [ Links ]

Sueyoshi, K., O. Hirata, and Y. Oji. 1997. Characterization of plasma membrane-bound Fe3+-chelate reductase from Fe-deficient and Fe-sufficient cucumber roots. Soil Sci. Plant Nutr. 43: 149-156. [ Links ]

Susín, S., A. Abadía, J. A. González-Reyes, J. J. Lucena, and J. Abadía. 1996. The pH requirement for in vivo activity of the iron-deficiency-induced “turbo” ferric chelate reductase (A comparison of the iron-deficiency-induced iron reductase activities of intact plants and isolated plasma membrane fractions in sugar beet). Plant Physiol. 110: 111-123. [ Links ]

Tagliavini, M., J. Abadía, A. Abadía, C. Tsipouridis, and B. Marangoni. 1997. Alternatives to Fe-chelates for overcoming fruit tree iron chlorosis Mediterranean countries. p. 112. In: 9th International Symposium on Iron Nutrition and Interactions in Plants. Hohenheim, Stuttgart, Germany. [ Links ]

Terry, N. and A. Zayed. 1995. Physiology and biochemistry of leaves under iron deficiency. pp. 283-294. In: J. Abadía (ed.). Iron nutrition in soils and plants. Kluwer Academic Publishers. Dordrecht, The Netherlands. [ Links ]

Young, T. F. and N. Terry. 1982. Transport of iron into leaves following iron resupply to iron-stressed sugar beet plants. J. Plant Nutr. 5: 1273-1283. [ Links ]

Zohlen, A. 2000. The use of 1, 10-phenanthroline in estimating metabolically active iron in plants. Commun. Soil Sci. Plant Anal. 31: 481-500. [ Links ]

Received: May 2012; Accepted: January 2013

* Autor responsable (

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License