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Morphological Changes inLeaves of Mexican LimeAffected by Iron ChlorosisRanferi Maldonado-Torres a , Jorge DionisioEtchevers-Barra b , Gabriel Alcántar-González b ,Jorge Rodriguez-Alcazar c & Maria Teresa Colinas-León da Departamento de Suelos , Universidad AutónomaChapingo , Chapingo, Méxicob Instituto de Recursos Naturales, Colegio dePostgraduados , Montecillo, Méxicoc Instituto de Recursos Genéticos y Productividad,Colegio de Postgraduados , Montecillo, Méxicod Departamento de Fitotecnia , UniversidadAutónoma Chapingo , Chapingo, MéxicoPublished online: 15 Aug 2006.

To cite this article: Ranferi Maldonado-Torres , Jorge Dionisio Etchevers-Barra ,Gabriel Alcántar-González , Jorge Rodriguez-Alcazar & Maria Teresa Colinas-León(2006) Morphological Changes in Leaves of Mexican Lime Affected by Iron Chlorosis,Journal of Plant Nutrition, 29:4, 615-628, DOI: 10.1080/01904160600564337

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Journal of Plant Nutrition, 29: 615–628, 2006

Copyright © Taylor & Francis Group, LLC

ISSN: 0190-4167 print / 1532-4087 online

DOI: 10.1080/01904160600564337

Morphological Changes in Leaves of Mexican LimeAffected by Iron Chlorosis

Ranferi Maldonado-Torres,1 Jorge Dionisio Etchevers-Barra,2

Gabriel Alcantar-Gonzalez,2 Jorge Rodriguez-Alcazar,3

and Maria Teresa Colinas-Leon4

1Departamento de Suelos, Universidad Autonoma Chapingo, Chapingo, Mexico2Instituto de Recursos Naturales, Colegio de Postgraduados, Montecillo, Mexico

3Instituto de Recursos Geneticos y Productividad, Colegio de Postgraduados,Montecillo, Mexico

4Departamento de Fitotecnia, Universidad Autonoma Chapingo,Chapingo, Mexico

ABSTRACT

Iron (Fe) chlorosis reduces the concentration of photosynthetic pigments, photosyn-thates, and crop yield. The effect of Fe chlorosis on leaf composition and cell structurewas evaluated in Mexican lime (Citrus aurantifolia) with different degrees of Fe chloro-sis. Iron chlorosis significantly reduced concentrations of chlorophylls a, b, and a + b,and caused thickening of leaves, due to the increase in palisade and spongy parenchymacells. The chloroplasts of the chlorotic and albino leaves showed a disorganized ul-trastructure; they had an elongated shape with disarrayed thylakoids, underdevelopedgrana, scarce starch granules, and hole-like folds in the thylakoid membranes. The accu-mulation of calcium oxalate crystals in the upper and lower sides of the epidermis, crystallength, and total crystal content increased with Fe chlorosis severity. The green leaves,in contrast, had chloroplasts with typical ultrastructure. The degree of Fe chlorosis in theleaves significantly affected the concentrations of potassium (K); Fe, manganese (Mn),Fe2+, and the phosphorus (P)/Fe and K/calcium (Ca) ratios.

Keywords: Citrus aurantifolia, mineral nutrition, Mexican lemon, iron deficiency,calcareous soil, alkaline soils, carbonates

Received 26 April 2004; accepted 31 May 2005.Address correspondence to Ranferi Maldonado-Torres, Departamento de Suelos,

Universidad Autonoma Chapingo, Chapingo, Mexico. E-mail: ranferimt@yahoo.com.mx

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INTRODUCTION

Although the earth’s crust is rich in total iron (Fe) (5%), and most soils haveadequate total amounts (0.7% to 5.5%), the fraction of available Fe is small(Romheld and Marschner, 1983; Loeppert, 1988). This is because the disso-lution and precipitation of Fe oxides [Fe(OH)3 + 3H+O Fe3+ + 3H2O] arecontrolled by factors in the soil that reduce the activity of Fe3+ in solution,causing a concentration (10−10 M) much smaller than that required (10−3 M)for proper plant nutrition (Lindsay and Schwab, 1982; Nedunchezhian et al.,1997). Plant Fe is essential for processes such as photosynthesis, respiration,nitrogen (N) fixation, and for DNA, chlorophyll, and hormone synthesis; Feis also a constituent of hemoproteins (cytochromes, catalase, and peroxidase)(Lobreaux and Briat, 1997). As a result, plants affected by Fe chlorosis suffersevere metabolic and structural disorders. Among them reduction in numberof cells per unit surface and disorganization of chloroplast structure, with noreduction in growth and size of the leaves (Terry and Abadıa, 1986). Chloro-plasts of beet leaves affected by Fe chlorosis are large in volume and have poorlaminar orientation, few or no grana, a large stroma, and a smaller number ofthylakoid membranes (Platt-Aloia et al., 1983). These changes have been re-lated to reduction in proteins, lipids and chlorophyll content within plant cells(Nishio et al., 1985; Chattopadhyay, 1988). The drop in the concentration ofchlorophyll is associated with a reduction in proteins, as both are joined bya non-covalent bond to form a pigment-protein complex that constitutes thethylakoid membrane (Bassi et al., 1990; Fodor et al., 1995). Leaves affectedby Fe chlorosis show changes in the concentration of nutrients. In this respect,Belkhodja et al. (1998) observed an increase in the concentration of potassium(K) and a high K/calcium (Ca) ratio in leaves and flowers of peach (Prunuspersica) trees deficient in Fe. These changes were attributed to an increase inthe excretion of hydrogen (H+) by plasma membrane ATPase in root cells,favoring the absorption of K (Marschner, 1986) and to the accumulation oforganic acids (malic and citric) that occurs in Fe-deficient organs (Welkie andMiller, 1993). According to Wallace and De Kock (1966), a low concentrationof Ca in chlorotic leaves could be because Fe blocks the activity of the enzymeaconitase, which regulates the isomerization of citric acid to isocitric acid inthe Krebs cycle. This blockage reduces protein synthesis and leads to free K+

accumulation. However, the high concentration of K+ relative to Ca2+ could bedue to a reduction in the synthesis of carbohydrates, which slows the movementof K+ from leaf to phloem vessels, causing immobility of K+ in chlorotic leavesand a reduction in the production of biomass (Hamze, 1983).

In general, the most visible symptom in plants affected by Fe deficiencyis intervenal yellowing of young leaves, known as Fe chlorosis. Iron chlorosishas been described in beans (Phaseolus vulgaris L.), soybean (Glycine max(L.)), cauliflower (Brassica oleracea Botrytis Group), broccoli (Brassica ol-eracea, Botrytis Group), cabbage (Brassica oleracea, Capitata Group), sugar

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beet (Beta vulgaris), spinach (Spinacia oleracea L.), maize (Zea mais L.), toma-toes (Licopersicon esculentum), cucumber (Cucumis sativus L.), green pepper(Capsicums), apple (Malus domestica), peach (Prunus persica), quince (Cy-donia oblonga), pear (Pyrus communis), plum (Prunus domestica L.), cherry(Prunus avium), almond (Prunus amygdalus), olive (Olea europaea), citrus(Citrus sp), kiwi (Actinidia Chinesis), etc., and even in ornamental plants suchas roses (Gonzalez-Vallejo et al., 1999). In fruit trees, Fe chlorosis is an im-portant economic consideration, as tree growth is reduced, flowering is scarce,and fruits are small and few, at times causing premature death of the tree (Sanzet al., 1992).

Metabolic alterations induced by Fe deficiency reduce growth and pro-duction; thus, the present study had the objective of evaluating morphologicaland nutritional changes in leaf tissue and alterations in cell chloroplasts ofleaves differentially affected by Fe chlorosis in order to better understand theseprocesses and search for ways to correct the disorder.

MATERIALS AND METHODS

Samples of leaves with three degrees of chlorosis (green or healthy, chlorotic,and albino) were collected from 20-year-old Mexican lime trees (Citrus au-rantifolia) grafted onto Citrus macrofila and planted in a calcareous soil in theregion of Apatzingan, Mexico. The trees were established at a distance of 10 ×10 m with a density of 100 tree ha−1 and average yield of 25 t ha−1.

Sections of fresh leaves were prepared for light and electron microscopes.For the first observation, the leaves were cut into 0.5 × 10 mm sections andfixed with FAA (10% formaldehyde, 5% acetic acid, 52% isopropilic acid, and33% water) for 12 h, then washed for 15 min. Sections were dehydrated in atissue-changer device (Fisher Tissuamaton). Before cutting in a microtome to athickness of 10 µ m, the preparation was treated with common paraffin at 60◦Cfor 6 h, then fixed and mounted in pure paraffin. Contrasts were highlighted withsaffranine and fixed green before adding the resin and a slide cover (Sass, 1968).Leaf thickness, palisade parenchyma and spongy thickness, cell diameter, longcrystals, crystals number in the adaxial and abaxial surface, total crystals, andcalcium oxalates were evaluated.

For the electron microscope, small pieces of fresh leaves were cut(<1.0 mm) and submerged in glutaraldehyde (3%) and a phosphate buffer0.1 M, pH = 7.0 (Karnovsky, 1963). The preparations were then washed withthe phosphate buffer solution, 0.1 M, pH = 7.0, and were postfixed for 2 hwith 1% osmium tetroxide. Washing with phosphate buffer was repeated. Thematerial was dehydrated with a series of increasingly pure (30%, 50%, 70%,80%, 96%, and 100%) ethyl alcohol applications. Finally, the preparations weresoaked in resin for fixation. They were then cut into ultra-thin sections with adiamond knife. The sections were contrasted with 4% uranyl acetate (Reynolds,

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1963), and six observations per treatment were made in a Zeiss M9 electronscanner microscope.

In one of the fresh leaf samples with a distinct degree of chlorosis, SPADunits were determined as an indirect measure of chlorophyll concentration. Toevaluate chlorophyll a and b quantitatively, a sample of 0.5 g of fresh leaves wastaken and homogenized with 40 mL of pure acetone. The extract was filteredthrough glass fiber and the residues were washed with 4 mL of acetone be-fore decanting into 100 mL of distilled water. The final extract consisted ofan 80% acetone solution, which was kept cold and protected from light. Theconcentration of chlorophyll a and b in these extracts was determined by mea-suring absorbancy at 645, 652, and 663 nm. The transformation of absorbancyto chlorophyll was performed following the specific equations proposed byBruisnma (1963).

To evaluate nutrient concentration, four- to seven-month-old leaves withno physical, chemical, or biological damage, and located in the fifth abaxialposition were collected from branches without fruit at the four cardinal pointsin trees with different grades of chlorosis in leaves. The sampled trees wereselected at random in a homogenous area. The leaves were placed in plastic bagsand taken to the lab in a portable cooler. Each sample was washed followingthe procedure proposed by Chapman (1960) and dried at 70◦C for 48 hoursin a forced-air oven before being ground in a stainless steel (20 screen) mill(Etchevers-Barra, 1988). The material was digested with 4 mL HNO3 and 2 mLHClO4 on an aluminum block heated gradually to 203◦C. During digestion, K,Ca, magnesium (Mg), Fe, zinc (Zn), manganese (Mn), copper (Cu), and boron(B) were determined by AES-ICP emission spectrophotometry (Varian). TotalN was determined by a Kjeldahl procedure with a salicylic modification toinclude NO−

3 and evaluated in distillation by vapor drag (Bremner, 1965). Thecalcium oxalate crystals were quantified using Pro-Plus software.

The data resulting from leaf-tissue analysis for concentration of chloro-phylls and morphological measurements of leaves and calcium-oxalate crystalswere subjected to an analysis of variance and tests of comparison of means(ANDEVA and TUKEY) using a significance of 5% in the Statistical AnalysisSystem and Microsoft Excel.

RESULTS AND DISCUSSION

Morphology of Leaves Affected by Iron Chlorosis

SPAD values for chlorophyll a, b, and a + b, determined in Mexican lime leaveswith different degrees of Fe chlorosis, are shown in Table 1. The differencesbetween these variables at various degrees of Fe chlorosis were significant.SPAD units and the concentration of chlorophylls a, b, and a + b decreased asseverity of Fe chlorosis increased.

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Table 1SPAD units and concentration of chlorophylls in Mexican limeleaves with differing degrees of iron chlorosis

Leaf appearance

Parameters Green Chlorotic Albino

SPAD units 58.28a∗ 28.98b 7.99cChlorophyll a (mg L−1) 6.12a 2.52b 1.11cChlorophyll b (mg L−1) 7.69a 3.43b 1.54cChlorophyll a + b (mg L−1) 15.84a 6.72b 2.63c

∗Different letters in the same line indicate significant differences(5%) according to the Tukey test.

These results coincide with those reported in the literature (Fan and Faust,1984; Abadıa et al., 1991; Morales et al., 1991). In leaves of sugar beet (Betavulgaris L.) affected by Fe chlorosis, a reduction in the concentration of photo-synthetic pigments and of components in the thylakoid membranes was found(Morales et al., 1991). In peach (Prunus) spp, reduction of 79% in the quantityof chlorophylls and 62% in quantity of carotenoids was observed, significantlyreducing net photosynthesis (Abadıa et al., 1991). Fan and Faust (1984) found areduction of 75% in net photosynthe thesis in leaves of apple (Malus domesticaL.) affected by Fe chlorosis compared with healthy leaves. It is speculated thatthe cause of this reduction is the control Fe has over the synthesis of chloro-phyll through aminolevulinic acid, protophyrinogen, and protochlorophyllide(Marschner, 1986). In addition, Fe participates in the synthesis of protopor-phyrine IX, the basic substance for the formation of heme groups, which areprecursors of chlorophylls (Bould et al., 1984). The effects of Fe deficiencyon leaf morphology with different degrees of chlorosis are shown in Figure 1.This figure shows images of cross-sections captured with a light microscopeand modified to contrast morphology and images of the chloroplast observedwith an electron-scanner microscope.

The data and tests of means for tissues, cells, and crystals visualized in thecross-section of leaves with different degrees of chlorosis are shown in Table 2.

The thickening of leaves with Fe deficiency was due to the longer andthicker cells of the palisade and spongy parenchyma. These results coincide withreports of thickening of root cortical cells in Fe-deficient plants (Landsberg,1996). In the case of roots, thickening is localized in the areas in which thepresence of Fe is reduced (Bell et al., 1988) and where proton extrusion occurs(Alcantara et al., 1991). Iron chlorosis produces morphological changes inplants in both the shoot and root, depending on the species. For example, in sun-flower (Helianthus annuus), a lengthening of cells and internodes in the shootwas observed (Romheld and Marschner, 1983), while in olive (Olea europea)shorter internodes and reduced leaf area were reported (Cordeiro, 1995).

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Table 2Test of means of morphological variables in leaves of Mexican limewith different degrees of affectation by iron chlorosis

Leaf appearance

Variables Green Chlorosis Albino

Leaf thickness (µm) 231a∗ 252a 258bPalisade parenchyma (µm) 74b 79ab 91aSpongy parenchyma (µm) 136b 159a 152abD. five cells (µm) 44b 53ab 60aL. crystals (µm) 30b 32ab 35aCrystals, Enves (number) 6b 10a 11aCrystals, Haz (number) 11a 12a 12aTotal crystals (number) 17b 22ab 23aOxalates (mg kg−1) 238.4b 298.7ab 324.9a

∗Different letters in the same line indicate significant differences(5%) according to the Tukey test.

As a result of the conditions in which the trees of this study developed,calcium oxalate crystals were observed in both the adaxial and abaxial sides ofthe leaves. The differences in crystal length, number of crystals in the abaxial,total crystals, and concentration of oxalates among the different degrees of Fechlorosis were significant. The length of the crystals and their numbers increasedin the back side as chlorosis increased in severity.

In preparations observed with the electron microscope, it was noted thatthere was a progressive deterioration of the chloroplasts as Fe chlorosis be-came more acute. The chloroplasts of Fe-deficient leaves, chlorotic and al-binos, showed a disorganized ultrastructure: lengthened shape and thylakoidspiled in disarray, underdeveloped grana, scarce starch granules, and hole-likefolds in the thylakoid membrane. In contrast, in green leaves a typical ultra-structure of the chloroplast was found: oblong shape, thylakoids piled in a widespace, well-developed grana, and presence of starch granules. Similar resultshave been reported for apple (Malus domestica) leaves affected by Fe chlorosis(Zhou et al., 1984). Insufficient Fe in sugar beets caused a significant reduc-tion in the number of thylakoid membranes, a form laminar with large volume,and poor orientation with few or no grana but large stroma (Platt-Aloia et al.,1983). Furthermore, Fe deficiency reduces the quantity of unsaturated fattyacids, and saturated fatty acids predominate in the constitution of the thylakoidmembranes, making the structure more rigid (Abadıa, 1992).

In the epidermis of the upper and lower parts of Mexican lime leaves, therewas an accumulation of oxalate crystals, whose number and size increased asthe severity of Fe chlorosis increased (see Figure 1). The size of the crystals

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was similar to that reported by Webb et al. (1995) in leaves of beans (Phase-olus vulgaris) and native grape (Vitis labrusca L.). It is known that calciumoxalate crystals form in the roots, stems, leaves, flowers, fruit, and seeds ofplants (Franceschi and Horner, 1980) due to the transformation of productsof the photorespiratory cycle of glycolate and glyoxilate to oxalate and to thecatabolism of ascorbic acid, which produces oxalate, or treonic or tartaric acid(Franceschi and Loewus, 1995).

These crystals are formed in response to an excess of Ca and to compensatefor the alkalization of the cytoplasm (Nakata and McConn, 2000). The crystalscan represent 6.3% of the leaf dry weight (Zindler-Frank, 1976) and up to 90% ofthe total Ca in leaves (Fink, 1991). Calcium accumulated in the form of oxalatemaintains a low level of free Ca in the cytoplasm (less than 10−7M) to preventinterference in the Ca-dependent signals (Bush, 1995) in the energy metabolismof the phosphates (Kretsinger, 1977) and in microskeletal dynamics (Hepler,1994), but maintains a continuous supply of Ca for the metabolic process inthe plant. This process leads to the assumption that the high concentration ofleaf Ca, which is optimally 3.73%–5.73% Ca for Mexican lime growing incalcareous soils (Maldonado et al., 2001), is found accumulated and inactive inthe form of calcium oxalate.

Nutrient Status of Leaves Affected by Iron Chlorosis

The concentration of some essential elements, as well as critical P/Fe and K/Caratios, are presented in Table 3. Also included in this table is the comparison ofmeans of these parameters as a function of chlorosis severity.

With the exception of P concentration, the degree of Fe chlorosis in theleaves significantly affected the concentration of K, Fe, Mn, and Fe2+, andthe P/Fe and K/Ca ratios. The ranges of P, K, Fe, and Mn concentrations

Table 3Comparison of means (Tukey) of the nutrient composition in leaves of Mexican limewith different degrees of iron chlorosis

Nutrient composition

% mg kg Ratios

Degree of chlorosis P K Fe Mn Fe2+ P/Fe K/Ca

Green 0.17a∗ 1.09b 88.14a 64.35a 16.81a 19.45b 0.26bChlorotic 0.17a 1.14b 65.20b 39.05b 10.54b 27.28ab 0.29bAlbino 0.21a 1.99a 56.91b 58.03ab 6.64c 37.57a 0.57a

∗Different letters in the same line indicate significant differences (5%) according tothe Tukey test.

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reported as optimal for Mexican lime are 0.18%–0.25%, 1.28%–1.87%, 61%–115 mg · kg−1, and 37%–73% mg · kg−1, respectively (Maldonado et al., 2001).The concentration of P in both green and chlorotic leaves was slightly less thannormal, while in the albino leaves it was normal. The concentration of K ingreen and chlorotic leaves was lower than the optimal value, while in albinoleaves the concentration was higher. Iron concentrations in green and chloroticleaves were close to optimum, while in albino leaves they were slightly lower.The concentration of Mn varied significantly with chlorosis severity; however,it was within the optimum range.

The effect of Fe chlorosis on the mineral composition of leaves differsamong species and growing conditions of the crop (Abadıa et al., 1989). Abadıaet al. (1985) observed that the concentrations of P, Ca, Mg, Na, Fe, Mn, Zn, andCu in peach leaves were not significantly affected by the severity of Fe chlorosis,but the concentration of K and K/Ca and P/Fe ratios were. The authors (Abadıaet al., 1989) reported an increase in the concentrations of N, K, and Ca in pearleaves when severity of Fe chlorosis increased, while the K/Ca ratio was notaffected. Concentrations of P, Mg, Zn, and Cu did not change, and Mn decreased.In leaves and flowers of peach trees deficient in Fe, Belkhodja et al. (1998) foundthat, as a consequence of Fe chlorosis, there was an increase in the concentrationof K and an increase in the K/Ca ratio, which were attributed to greater excretionof H+ by the ATPase of the plasmalemma of root cells that favor the exchangeand absorption of K (Marschner, 1986). However, Welkie and Miller (1993) alsorelate the accumulation of K to the increase and accumulation of organic acids(malic and citric) that are present in Fe-deficient organs. Wallace and De Kock(1966) thought that the high K/Ca ratio was caused by the low concentration ofCa in chlorotic leaves because Fe controls the activity of the enzyme aconitase,which regulates the isomerization of citric acid to isocitric acid in the Krebscycle, reducing protein synthesis and promoting the formation of free K+.

Hamze et al. (1980) studied in citrus rootstock the concept that chloroticplants have insufficient K+ absorption and an excessive translocation of Ca2+.However, the high concentration of K+ relative to Ca2+ is known to be dueto a reduction in the synthesis of carbohydrates. This reduction slows downthe movement of K+ from the leaf to the phloem vessels, and even stops it inchlorotic leaves, and also reduces biomass production (Hamze, 1983).

The results of this study coincide with those of Abadıa et al. (1989),Belkhodja et al. (1998), Hamze et al. (1980), Hamze (1983), Marschner (1986),Wallace and De Kock (1966), and Welkie and Miller (1993) in that the con-centration of Ca2+ in affected leaves was not modified by Fe chlorosis, butabsorption and accumulation of K+ increased, resulting in a higher K/Ca ratio,1.25; 1.50, in chlorotic leaves.

Although chlorosis did not significantly affect the concentration of P, aslight tendency to increase was observed under more acute Fe chlorosis. Incontrast, the concentration of Fe was significantly affected, and this inverserelationship caused a significant increase in the P/Fe ratio. Similar results have

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been reported by Abadıa et al. (1985), Belkhodja et al. (1998), and Dong (1987),and Ji et al. (1985). The values reported vary between 20 and 27 for healthyleaves and between 25 and 37 for chlorotic leaves.

The concentration of Mn tended to decrease as an effect of Fe chloro-sis. However, the results reported by Belkhodja et al. (1998) indicate that innon-chlorotic peach leaves the concentration of Mn was 37 mg kg−1, and inchlorotic leaves it increased to 40 mg kg−1.

In the case of Fe, similar results have been found in chlorotic peach and pearleaves, with Fe concentrations of 84.2 and 86.3 mg kg−1, respectively (Moraleset al., 1998). These results show that the plants affected by Fe chlorosis havesimilar Fe levels, which are higher than those of normal plants (Abadıa et al.,1985). This excess suggests that Fe may be accumulated in a non-availableform in chlorotic leaves (Marschner et al., 1986) and, therefore, Fe content maynot be the best indicator of Fe chlorosis. Therefore, it has been proposed thatthe nutrient state of chlorotic plants should be evaluated by the quantificationof active Fe (Katyal and Sharma, 1980; Zohlen, 2000). Iron active (Fe2+) isfundamental in the synthesis of protoporphirine IX, the precursor of chloro-phylls; the close relationship of Fe2+ to chlorophylls and chlorosis makes thedetermination of Fe2+ a good indicator of the nutrient status of crops (Katyaland Sharma, 1980).

CONCLUSIONS

Iron chlorosis, caused by the lack of assimilable Fe in the soil, promoted mor-phological, cellular, and ultracellular changes in leaves of Mexican lime.

Morphological and cellular changes were associated with an increase inthe thickness and size of the leaf and with the thickness and size of palisadeand spongy parenchyma cells. At the ultracellular level, it was found that thechloroplasts from chlorotic and albino leaves had an elongated shape, withthylakoids piled in a disorderly manner, underdeveloped grana, scarce starchgranules, and hole-like folds in the thylakoid membrane; in short, there was ageneral disorganization within the chloroplast.

The severity of Fe chlorosis in Mexican lime leaves was associated with asignificant decrease in the concentrations of chlorophyll a, b, and a + b with theaccumulation, length, and total content of calcium oxalate crystals in the upperand lower epidermis, and with an increase in the concentrations of K, total Fe,Mn, Fe2+, and P/Fe, and K/Ca ratios.

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