Effects of Nitric Oxide on Iron-Deficiency Stress Alleviation of Peanut

The aim of this research was to study the role of nitric oxide (NO) in alleviating iron deficiency induced chlorosis of peanut (Arachis hypogaea L.). For this study, sodium nitroprusside (SNP) was used to supply NO for hydroponic peanut plants. After 18 days, the peanut seedlings growing without iron exhibited significant leaf interveinal chlorosis, and this iron-deficiency induced symptom was completely prevented by NO. An increased content of chlorophyll and active iron was observed in NO-treated young leaves, suggesting an improvement of iron availability in plants. In addition, the improved rhizosphere acidification and increased secretion of organic acids by root in NO-treated plants suggesting that NO is effective in modulating iron uptake and transport inside the peanut plants. Furthermore, NO treatment alleviated the increased accumulation of superoxide anion (O₂^??) and malondialdehyde (MDA), and modulated the antioxidant enzymes. However, the SNP with a prior sunlight treatment that does not release NO had no significant effect on the chlorophyll levels in iron-deficient plants. Therefore, these results support a physiological action of NO on the availability, uptake and transport of iron in the plant.     

Effects of exogenous SA supplied with different approaches on growth,chlorophyll content and antioxidant enzymes of peanut growing on calcareous soil

To assess the role of salicylic acid (SA) supplied with 5 approaches in alleviating chlorosis induced by iron (Fe) deficiency in peanut plants growing on calcareous soil, SA was supplied as soil incorporation, making slow-release particles, seed soaking, irrigation and foliar application. SA application, particularly, SA supplied by slow release particles, dramatically increased growth parameters, yield and quality of peanut, and increased Fe concentration in peanut grain. Meanwhile, SA application increased the H⁺-ATPase activity, reduced pH of soil, increased Fe³⁺-Chelate Reductase (FCR) activity in roots, and increased Fe concentration in roots. Furthermore, SA increased active Fe content and increased chlorophyll content. In addition, SA improved enzymes activities containing superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and protected Fe deficiency induced oxidative stress. Therefore, SA has a good effect on alleviating chlorosis induced by Fe deficiency on calcareous soil. However, in the 5 SA supplied approaches, foliar application and making slow release particles were more effective.     

Effects of root addition and foliar application of nitric oxide and salicylic acid in alleviating iron deficiency induced chlorosis of peanut seedlings

Nitric oxide (NO) and salicylic acid (SA) are two important signaling molecules, which could alleviate chlorosis of peanut under iron (Fe) deficiency. Here, we further investigated the mechanism of different combinations of sodium nitroprusside (SNP, a nitric oxide donor) and SA supplying in alleviation Fe deficiency symptoms and selected which is the best combination. Thus, peanut was cultivated in hydroponic culture under iron limiting condition with different combinations of SNP and SA application. After 21 days, Fe deficiency significantly inhibited peanut growth, decreased soluble Fe concentration and chlorophyll contents, and disturbed ionic homeostasis. In addition, the content of reactive oxygen species (ROS) and malondialdehyde (MDA) concentration increased, which led the lipid peroxidation. Application of SNP and SA significantly changed Fe trafficking in cells and organs, which increased Fe uptake from nutrient solution, and transport from root to shoot, enhanced the activity of ferric-chelate reductase (FCR), that increased the available Fe in cell organelles, and the active Fe, chlorophyll contents in leaves. Furthermore, ameliorated the inhibition of calcium (Ca), magnesium (Mg) and zinc (Zn) uptake and promoted plant growth in Fe deficiency. At the same time, it increased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) to protect the plasmolemma from peroxidation. Results demonstrated that different combinations of SNP and SA application could alleviate the chlorosis of peanut in Fe deficiency by various mechanisms. Such as increased the available Fe and chlorophyll concentrations in leaves, improved the activities of antioxidant enzymes and modulated the mineral elements balance and so on. Foliar application of SNP and SA is the best to protect leaves while directly adding them into nutrient solution is the best to protect roots. These results also indicated that the effects of SNP and SA supplying together to leaves or roots are better than respectively adding to roots and spraying to leaves. The best combination is foliar application of SNP and SA.     

Iron treatment of lime‐induced chlorosis: Implications for chlorophyll,Fe2 +, Fe3 + and K+ in leaves 1

This study addressed some complementary aspects related to plant Fe nutrition. A field and a greenhouse experiment were conducted to monitor changes in chlorophyll, Fe³⁺, Fe²⁺, Ca²⁺ and K⁺ along with the progressive evolution of lime‐induced chlorosis, and following soil (Fe‐EDDHA, Fe‐EDTA, Fe‐DTPA, DTPA) and foliar (Fe‐EDDHA, FeSO₄, “Fe‐Metalosate") treatments, in a chlorosis‐susceptible ornamental plant, Hydrangea macrophylla, over a year's growing period. Though soil Fe‐EDDHA was the most effective compound in alleviating chlorosis symptoms, it became less so with time and was only partly effective as a foliar spray. Leaf analysis showed that as chlorosis intensified and chlorophyll content decreased, phenanthroline ‐ Fe (Fe²⁺) decreased with corresponding increases in total iron (Fe³⁺) and K⁺ concentrations. The reliability of these chlorosis‐indicators was confirmed as the reverse changes occurred upon chlorosis plant recovery.     

Effect of application of exogenous nitric oxide at different critical growth stages in alleviating Fe deficiency chlorosis of peanut growing in calcareous soil

This study was to investigate peanut response to application of nitric oxide (NO) at different growth stages and the effects of NO application on peanut yield and quality in calcareous soil. Sodium nitroprusside (SNP, a NO donor) solution was poured into calcareous soil at sowing, seedling, flowering, and podding stages, respectively, or at each aforesaid critical stage. Results showed that NO application increased the content of active Fe and leaf chlorophyll, which improved the photosynthesis of peanut; enhanced the ability of resistance to oxidative stress by decreased the accumulation of O₂^??, H₂O₂, and MDA and increased the activity of antioxidant enzymes. Nitric oxide increased the content of soil available Fe and root FCR activity, which can promote peanut absorb more Fe from the calcareous soil. What's more, peanut plants may pump a large amount of H⁺ from root cell membrane to consume in neutralization of HCO₃^?, and decrease the pH in apoplast, cytoplasm, and xylem, finally balance the mineral elements (Fe, Ca, Mg, Zn, and Cu) uptake and distribution. These results indicated that NO could improve peanut growth and development, increase peanut yield and quality. Furthermore, the application of NO at sowing or seedling stage did the most obvious effect on alleviating chlorosis of peanut in calcareous soil.     

Effect of potassium salts on alleviation of lime‐induced chlorosis in soybean 1

Studies were conducted to determine the efficacy of K salts in alleviating lime‐induced chlorosis. Greenhouse studies using a Gibbon silt loam [fine‐silty, mixed (calcareous), mesic Typic Haplaquoll] and a 1: 1 mixture of Gibbon soil and washed sand were conducted with KCl, KNO₃, K₂SO₄, K₂HPO₄, or KHCO₃ applied at rates of 0, 250, and 500 mg K/kg soil. An FeEDDHA treatment was included for comparison. Similar studies were conducted at two field sites known to produce lime‐induced chlorosis. Potassium salts were applied at 0, 20, and 40 g K/m of row. In the greenhouse, plants treated with KCl, KNO₃, and K₂SO₄ on Gibbon soil were less chlorotic than controls or plants treated with K₂HPO₄, or KHCO₃. No K treatment totally alleviated chlorosis except FeEDDHA. Chlorophyll correlated positively with chlorosis rating. No relationship was found between leaf Fe uptake and chlorosis. Plants grown in soil/sand exhibited no chlorosis and had lower Fe uptake than plants grown in Gibbon soil. Thus chlorosis was not due strictly to low soil‐Fe availability or inadequate Fe uptake. Bicarbonate in the soil solutions of both growth media treated with KCl was lower than controls which may explain the reduced chlorosis associated with this treatment.

One field site showed positive effects of K treatments on chlorosis rating, chlorophyll concentration, and seed yield. No treatment was as effective as FeEDDHA in influencing plant growth or yield. Total leaf Fe concentration was unrelated to leaf chlorophyll concentration. Inorganic cation/anion ratios in the plant were from 4.4–8.4 which could cause net H⁺ efflux by the plant and alkalinization of plant tissues. One possibility is that H⁺ efflux solubilizes P in the rhizosphere, which after uptake could immobilize Fe in the plant. Application of KCl, KNO₃, and K₂SO₄ generally lowered HCO₃ content of the upper 15 cm of both soils. High bicarbonate could increase rhizosphere P availability and increase immobilization of Fe in the plant.     

Soil Properties Influencing Iron Chlorosis in Grapevines Grown in the Montilla‐Moriles Area,Southern Spain

Abstract

Iron (Fe) chlorosis, an Fe deficiency commonly observed in grapevines cultivated on calcareous soils, generally inhibits plant growth and decreases yield. The objective of this research was to relate the incidence of Fe chlorosis in vines of the Montilla‐Moriles area, southern Spain, to indigenous soil properties. Thirty‐five grapevines (V. vinífera L. cv. Pedro Ximenez grafted on V. berlandieri×V. rupestris 110 Ritcher) showing different degree of Fe chlorosis were selected from 13 vineyards. The leaf chlorophyll concentration (estimated by the SPAD value measured with a Minolta meter) was positively correlated with the contents in different soil Fe forms but not with alkalinity‐related soil properties (pH, calcium carbonate equivalent, and active lime). The acid NH₄ oxalate‐extractable Fe (Fe_o) was the most useful simple variable to predict the occurrence of Fe chlorosis. A Fe_o/active lime ratio of 25×10^–4 was found to be useful to class soils into two groups according to the probability of inducing Fe chlorosis.     

Glutathione foliar fertilisation prevents lime‐induced iron chlorosis in soil grown Medicago scutellata

It has been proposed that glutathione can relieve the effects of Fe deficiency. This study tested the effects of glutathione foliar treatments to prevent Fe chlorosis, using as positive controls soil and foliar Fe fertilisation. Medicago scutellata plants were grown in soil (5.7% CaCO₃) supplemented or not with 4 and 8% CaCO₃. Two Fe(III)‐EDDHA soil treatments (5 and 10 mg Fe kg^?1), and three foliar treatments (three applications each of 2.14 mM Fe(III)‐EDDHA, 1 mM glutathione, and the previous two combined) were applied. Measurements include leaf chlorophyll and Fe concentrations, biomass, leaf enzymatic and non‐enzymatic antioxidant systems and carboxylates. The addition of CaCO₃ caused typical Fe deficiency symptoms, including changes in chlorophyll, Fe, antioxidant systems and carboxylates, which were prevented by soil and foliar Fe fertilisation. The foliar treatment with glutathione also led to higher chlorophyll, leaf extractable Fe and root Fe, as well as decreases in some antioxidant systems, whereas leaf Fe concentrations decreased. The combined foliar application of glutathione and Fe was even more efficient in preventing chlorosis. Including glutathione in foliar fertilisation programs should be considered as an option for Fe chlorosis prevention, especially when relatively large leaf total Fe concentrations occur in the so called chlorosis paradox.     

EFFECTIVENESS OF DIFFERENT FOLIAR IRON APPLICATIONS TO CONTROL IRON CHLOROSIS IN ORANGE TREES GROWN ON A CALCAREOUS SOIL

The effectiveness on controlling Fe chlorosis in orange trees grown on calcareous soils was tested. The treatments were Fe(II) sulfate (500 mg Fe L^?1), sulfuric acid (0.5 mM H₂SO₄), Fe(III)-chelate (Hampiron 654 GS, 120 mg Fe L^?1) and distilled water as a control. A non-ionic wetting agent was used in all treatments. The use of frequent foliar sprays alleviated Fe chlorosis in orange trees. Sprays of Fe(II) sulfate increased the concentrations of chlorophyll, Fe and zinc in leaves and improved fruit size and quality compared to fruits of control trees. Sprays of Fe(III)-chelate also increased leaf chlorophyll and Fe concentrations and improved fruit quality, but did not increase fruit size. Sprays of sulfuric acid alone slightly increased leaf chlorophyll and Fe concentrations, without improving fruit size and quality. These results suggest that foliar sprays with Fe could help to avoid yield and quality losses caused by Fe chlorosis in citrus orchards. Furthermore, these treatments could be done with relatively cheap materials such as solutions containing Fe(II) sulfate.     

Mechanisms of exogenous nitric oxide and 24-epibrassinolide alleviating chlorosis of peanut plants under iron deficiency

Iron(Fe) is a crucial transition metal for all living organisms including plants; however, Fe deficiency frequently occurs in plant because only a small portion of Fe is bioavailable in soil in recent years. To cope with Fe deficiency, plants have evolved a wide range of adaptive responses from changes in morphology to altered physiology. To understand the role of nitric oxide(NO) and 24-epibrassinolide(EBR) in alleviating chlorosis induced by Fe deficiency in peanut(Arachis hypogaea L.) plants, we determined the concentration of chlorophylls, the activation, uptake, and translocation of Fe, the activities of key enzymes, such as ferric-chelate reductase(FCR),proton-translocating adenosine triphosphatase(H~+-ATPase), and antioxidant enzymes, and the accumulation of reactive oxygen species(ROS) and malondialdehyde(MDA) of peanut plants under Fe sufficiency(100 μmol L~(-1)ethylenediaminetetraacetic acid(EDTA)-Fe) and Fe deficiency(0 μmol L~(-1)EDTA-Fe). We also investigated the production of NO in peanut plants subjected to Fe deficiency with foliar application of sodium nitroprusside(SNP), a donor of NO, and/or EBR. The results showed that Fe deficiency resulted in severe chlorosis and oxidative stress, significantly decreased the concentration of chlorophylls and active Fe, and significantly increased NO production. Foliar application of NO and/or EBR increased the activity of antioxidant enzymes, superoxide dismutase,peroxidase, and catalase, and decreased the ROS and MDA concentrations, thus enhancing the resistance of plants to oxidative stress.Application of NO also significantly increased Fe translocation from the roots to the shoots and enhanced the transfer of Fe from the cell wall fraction to the cell organelle and soluble fractions. Consequently, the concentrations of available Fe and chlorophylls in the leaves were elevated. Furthermore, the activities of H~+-ATPase and FCR were enhanced in the Fe-deficient plants. Simultaneously,there was a significant increase in NO production, especially in the plants that received NO, regardless of Fe supply. These suggest that NO or EBR, and, especially, their combination are effective in alleviating plant chlorosis induced by Fe deficiency.     

CHLOROSIS IN WILD PLANTS: IS IT A SIGN OF IRON DEFICIENCY?

ABSTRACT

Chlorosis in crops grown on calcareous soil is mainly due to iron (Fe) deficiency and can be alleviated by leaf application of soluble Fe²⁺ or diluted acids. Whether chlorosis in indigenous plants forced to grow on a calcareous soil is also caused by Fe deficiency has, however, not been demonstrated. Veronica officinalis, a widespread calcifuge plant in Central and Northern Europe, was cultivated in two experiments on acid and calcareous soils. As phosphorus (P) deficiency is one of the major causes of the inability of many calcifuges to grow on calcareous soil we added phosphate to half of the soils. Leaves in pots with the unfertilized and the P-fertilized soil, respectively, were either sprayed with FeSO₄ solution or left unsprayed. Total Fe, P, and manganese (Mn) in leaves and roots and N remaining in the soil after the experiment were determined. In a second experiment, no P was added. Leaves were either sprayed with FeSO₄ or with H₂SO₄ of the same pH as the FeSO₄ solution. Degree of chlorosis and Fe content in leaves were determined. Calcareous soil grown plants suffered from chlorosis, which was even more pronounced in the soils supplied with P. Newly produced leaves were green with Fe spray but leaves that were chlorotic before the onset of spraying did not totally recover. H₂SO₄ spray even increased chlorosis. This demonstrated that chlorosis was due to Fe deficiency. As total leaf Fe was similar on acid and calcareous soil, it was a physiological Fe deficiency, caused by leaf tissue immobilization in a form that was not metabolically “active”. Iron in the leaves was also extracted by 1,10-phenanthroline, an Fe chelator. In both experiments, significant differences between leaves from acid and calcareous soil were found in 1,10-phenanthroline extractable Fe but not in total leaf Fe, when calculated on a dry weight basis. Differences in 1,10-phenanthroline extractable Fe were more pronounced when calculated per unit dry weight than calculated per leaf area, whereas the opposite condition was valid for total leaf Fe.     

Activities of Iron‐Containing Enzymes in Leaves of Two Tomato Genotypes Differing in Their Resistance to Fe Chlorosis

Abstract

Two tomato (Lycopersicon esculentum Mill., cvs. Pakmor and Target) genotypes differing in resistance to iron (Fe) deficiency were grown in nutrient solution under controlled environmental conditions over 50 days to study the relationships between severity of leaf chlorosis, total concentration of Fe, and activities of Fe‐containing enzymes in leaves. The activities of Fe‐containing enzymes ascorbate peroxidase, catalase, and guaiacol peroxidase, and additionaly the activity of glutathione reductase, an enzyme that does not contain Fe, were measured. Plants were supplied with 2 × 10^?7 M (Fe deficient) and 10^?4 M (Fe sufficient) FeEDTA, respectively. Leaf chlorosis appeared more rapidly and severely in Target (Fe deficiency senstive genotype) than Pakmor (Fe deficiency resistant genotype). On day 50, Pakmor had 2‐fold more chlorophyll than Target under Fe deficiency, while at adequate supply of Fe the two genotypes were very similar in chlorophyll concentration. Despite distinct differences in development of leaf chlorosis and chlorophyll concentrations, Pakmor and Target were very similar in concentrations of total Fe under Fe deficiency. In contrast to Fe concentration, activities of Fe‐containing enzymes were closely related to the severity of leaf chlorosis. The Fe‐containing enzymes studied, especially catalase, showed a close relationship with the concentration of chlorophyll and thus differential sensitivity of tomato genotypes to Fe deficiency. Glutathione reductase did not show relationship between Fe deficiency chlorosis and enzyme activity. The results confirm that measurement of Fe‐containing enzymes in leaves is more reliable than the total concentration of Fe for characterization of Fe nutritional status of plants and for assessing genotypical differences in resistance to Fe deficiency. It appears that Fe deficiency‐resistant genotype contains more physiologically available Fe in tissues than the genotype with high sensitivity to Fe deficiency.     

Biochemical Responses to True and Bicarbonate-Induced Iron Deficiency in Grapevine Genotypes

ABSTRACT

Biochemical responses to direct or bicarbonate-induced iron (Fe) deficiency were compared in two Tunisian native grapevine varieties, Khamri (tolerant) and Balta4 (sensitive), and a tolerant rootstock, 140Ru. Woody cuttings of each genotype were irrigated with a nutrient solution containing one of the following: 20 μM Fe (control), 1 μM Fe (direct Fe-deficiency), or 20 μM Fe + 10 mM HCO₃ ^? (indirect bicarbonate-induced Fe-deficiency). Under direct Fe-deficient conditions, lower leaf chlorosis score and higher chlorophyll and leaf Fe contents were found in Khamri and 140Ru compared with Balta4. Moreover, indirect Fe deficiency caused similar effects on these parameters, which were more pronounced in Balta4. Both tolerant genotypes, Khamri and 140Ru, showed higher roots-acidification capacity and phenol release under the direct Fe deficiency compared with the bicarbonate-induced condition. In the sensitive variety, Balta4, no significant changes were found between the control and Fe-deficient plants. Root Fe(III)-reductase activity was strongly stimulated by both types of Fe deficiency in Khamri and 140Ru, and displayed no significant changes in Balta4. In the three genotypes, root and leaf activities of two Fe-containing enzymes, catalase and guaiacol peroxidase, were significantly affected under Fe deficiency (either direct or induced), though to a greater extent in the sensitive variety, Balta4. The latter also displayed higher leaf malonyldialdehyde (MDA) content, traducing an important membrane lipid peroxidation.     

Effects of Exogenous Salicylic Acid and Nitric Oxide on Peanut Seedlings Growth under Iron Deficiency

Salicylic acid (SA) and nitric oxide (NO), which are known as important signaling molecules in plants, could be promising compounds for the reduction in stress sensitivity. The aim of the present work was to study the physiological changes in peanut (Arachis hypogaea L.) seedlings grown in growth medium that contained 0.1 mM SA, 0.25 mM sodium nitroprusside (SNP, a NO donor), or in full (SA+SNP) or half [1/2 (SA+SNP)] combined strengths under iron (Fe) deficiency. After 21 days of treatment, Fe deficiency significantly inhibited peanut plant growth, destroyed photosynthetic system, and caused oxidative damages. Addition of SA, SNP, and 1/2 (SA+SNP), especially SA+SNP, alleviated the stress, increased the contents of chlorophylls, and promoted plant growth. They improved Fe uptake, transport, and availability in peanut plants by increasing the activities of H⁺-ATPase and ferric chelate reductase (FCR), and promoting Fe translocation from cell wall to cell organelle and soluble fraction in leaves. Furthermore, they also effectively mitigated oxidative damages by increasing the activities of antioxidant enzymes in peanut leaves and roots. The results from the present study indicate that application of SA, SNP, or 1/2 (SA+SNP) can overcome the adverse effect of Fe deficiency, but the combined application of SA+SNP is more effective in alleviating Fe deficiency stress.     

Root and Leaf Ferric Chelate Reductase Activity in Pond Apple and Soursop

Abstract

Root and leaf ferric chelate reductase (FCR) activity in Annona glabra L. (pond apple), native to subtropical wetland habitats and Annona muricata L. (soursop), native to nonwetland tropical habitats, was determined under iron (Fe)-sufficient and Fe-deficient conditions. One-year-old seedlings of each species were grown with 2, 22.5, or 45 µM Fe in a nutrient solution. The degree of tolerance of Fe deficiency was evaluated by determining root and leaf FCR activity, leaf chlorophyll index, Fe concentration in recently mature leaves, and plant growth. Root FCR activity was generally lower in soursop than in pond apple. Eighty days after plants were put in nutrient solutions, leaf FCR activity of each species was lower in plants grown with low Fe concentrations (2 µM) than in plants grown with high (22.5 or 45 µM) Fe concentrations in the nutrient solution. Leaves of pond apple grown without Fe became chlorotic within 6 weeks. The Fe level in the nutrient solution had no effect on fresh and dry weights of soursop. Lack of Fe decreased the leaf chlorophyll index and Fe concentration in recently matured leaves less in soursop than in pond apple. The rapid development of leaf chlorosis in low Fe conditions and low root and leaf FCR activities of pond apple are probably related to its native origin in wetland areas, where there is sufficient soluble Fe for adequate plant growth and development. The higher leaf FCR activity and slower growth rate of soursop compared to pond apple may explain why soursop did not exhibit leaf chlorosis even under low Fe conditions.     

Influences of ultra‐violet (UV)‐blue light radiation on the growth of cotton. I. Effect on iron nutrition and iron stress response

Cool white fluorescent (CWF) light reduces Fe³⁺ to Fe²⁺ while low pressure sodium (LPS) light does not. Cotton plants grown under CWF light are green, while those yrown under LPS light develop a chlorosis very similar to the chlorosis that develops when the plants are deficient in iron (Fe). It could be that CWF light (which has ultra violet) makes iron more available for plant use by maintaining more Fe²⁺ in the plant. Two of the factors commonly induced by Fe‐stress in dicotyledonous plants‐‐hydroyen ions and reductants released by the roots‐‐were measured as indicators of the Fe‐deficiency stress response mechanism in M8 cotton.

The plants were grown under LPS and CWF light in nutrient solutions containing either NO₃‐N or NH₄‐N as the source of nitrogen, and also in a fertilized alkaline soil. Leaf chlorophyll concentration varied significantly in plants grown under the two light sources as follows: CWF+Fe > LPS+Fe > CWF‐Fe ≥ LPS‐Fe. The leaf nitrate and root Fe concentrations were significantly greater and leaf Fe was generally lower in plants grown under LPS than CWF light. Hydrogen ions were extruded by Fe‐deficiency stressed roots grown under either LPS or CWF light, but “reductants”; were extruded only by the plants grown under CWF light. In tests demonstrating the ability of light to reduce Fe³⁺ to Fe²⁺ in solutions, enough ultra violet penetrated the chlorotic leaf of LPS yrown plants to reduce some Fe³⁺ in a beaker below, but no reduction was evident through a yreen CWF grown leaf.

The chlorosis that developed in these cotton plants appeared to be induced by a response to the source of liyht and not by the fertilizer added. It seems possible that ultra violet liyht could affect the reduction of Fe³⁺ to Fe²⁺ in leaves and thus control the availability of this iron to biological systems requiring iron in the plant.     

Determination of iron chlorosis with extractable iron analysis in peach leaves

Iron (Fe) though indispensable for the biosynthesis of chlorophyll, but its total content in the plant was not associated with the occurrence of chlorosis. Iron, which is the ferrous‐iron (Fe²⁺) form—termed “active”; Fe— and extracted with weak acids and some chelating agents, has been closely related to Fe chlorosis. In this study, three different methods were tested in order to determine suitable methods for extractable‐Fe analysis in a Dixired peach cultivar. In the first two methods, o‐phenantroline (o‐Ph) and 1N hydrochloric acid (HCl) were used to extract Fe²⁺ from fresh leaves. In the third method, 1N HCl were used as an extractant on dried leaf samples. The relationship between chlorophyll content of the leaves and Fe extracted by the three methods, was statistically significant. Hydrochloric acid extraction with dried leaves which gave the highest significant correlation (r = 0.930) with chlorophyll content, can be used for the determination of Fe²⁺ ("active”; Fe) status in peach trees.     

Comparative investigation on the susceptibility of faba bean (Vicia faba L.) and sunflower (Helianthus annuus L.) to iron chlorosis

Comparative physiological studies on iron (Fe) chlorosis of Vicia faba L. and Helianthus annuus L. were carried out. High internal Fe contents in Vicia cotyledons (16–37 μg) were completely used for plant growth and Fe chlorosis was not inducible by the application of nitrate (with or without bicarbonate). In Helianthus, low quantities of Fe in the seeds (4 μg) were insufficient for normal growth and without Fe in the nutrient solution, Fe chlorosis was obtained in all treatments. This chlorosis was an absolute Fe deficiency. Also, the treatment with 1 μM Fe in the nutrient solution and nitrate (with or without bicarbonate) led to severe chlorotic symptoms associated with low leaf Fe concentrations and high Fe concentrations in the roots. In contrast, Helianthus grown with NH₄NO₃ and 1 μM Fe had green leaves and high leaf Fe concentrations. However, with NO₃ supply (with or without bicarbonate), Fe translocation from the roots to the upper plant parts was restricted and leaves were chlorotic. Chlorotic and green sunflower leaves may have the same Fe concentrations, leaf Fe concentration being dependent on Fe translocation into the leaf at the various pH levels in the nutrient solution. At low external pH levels (controlled conditions) more Fe was translocated into the leaf leading to similar leaf Fe concentrations with higher chlorophyll concentrations (NH₄NO₃) and with lower chlorophyll concentrations (NO₃). This indicates a lower utilization of leaf Fe of NO₃ grown sunflower plants. Utilization of Fe in faba bean leaves is presumably higher than in sunflower leaves. In Vicia xylem sap pH was not affected by nitrate. In contrast, the xylem sap pH in Helianthus was permanently increased by about 0.4 pH units when fed with nitrate (with or without bicarbonate) compared with NH₄NO₃ nutrition. The xylem sap pH is indicative of leaf apoplast pH. From our earlier work (Mengel et al., 1994; Kosegarten und Englisch, 1994) we therefore suppose that in Helianthus, Fe immobilization occurs in the leaf apoplast due to high pH levels when grown with nitrate (with or without bicarbonate).     

ADVERSE EFFECTS OF HUMIC SUBSTANCES FROM DIFFERENT ORIGIN ON LUPIN AS RELATED TO IRON SOURCES

Humic substances improve the efficiency of different iron (Fe) sources overcoming Fe deficiency chlorosis of plants. However, applied at high rates, they can promote negative effects on plants. The main objective of this work was to study the potential adverse effect of three humic acids from different origin when they were applied with two effective Fe sources for plants: Fe- ethylenediaminedihydroxyphenylacetic acid (EDDHA) and Vivianite. To this end, an experiment with lupin (Lupinus albus L.) was performed involving two factors: (i) Fe source, and (ii) humic substances from three different origin (composted cork, leonardite, and compost obtained from a mixture of olive husk with cotton gin trash) applied at 0, 0.1, and 0.5 g organic carbon (C) kg^?1 of growing media. At the rates used, humic substances promoted adverse effects on plant development, chlorophyll meter readings, and Fe content in lupin grown in calcareous media. Overall, the effect on dry matter and Fe content in plants was more relevant when Fe was supplied with Vivianite, the effect on chlorophyll meter readings being more significant when Fe was applied as Fe-EDDHA. Differences were also observed depending on the source of humic substances, those from leonardite promoting the greatest decrease in dry matter in roots and shoots. These humic substances possessed the highest values of spectroscopy index for aromaticity (A₂₅₄ ). On the other hand, the application of humic substances from olive husk compost, which exhibited the lower aromaticity index, resulted in the smallest decrease in dry matter production and chlorophyll meter readings. Dry matter in roots decreased logarithmically with increased values of the estimates of the amounts of aromatic compounds accumulated in the growing media (R² = 0.92; P < 0.01) with Vivianite as Fe source. Thus, the effects decreasing dry matter production, particularly in roots, and chlorophyll meter readings can be ascribed at least partially to the presence of phytotoxic aromatic compounds in humic substances.     

ROLE OF SALICYLIC ACID IN REGULATION OF CADMIUM TOXICITY IN WHEAT (TRITICUM AESTIVUM L.)

The present study is focused on the possible mediatory role of salicylic acid (SA) under cadmium (Cd⁺²) toxicity. Cadmium treatments resulted in the inhibition of root dry biomass, root elongation and increased Cd accumulation in roots. Pretreatment of seeds with SA (500 μM) for 20 hrs resulted in protection against Cd, increased root dry biomass, root elongation and minimal accumulation of cadmium. Cadmium chloride (CdCl₂; 0.0, 100, 400 and 1000 μM) produced a significant inhibition in chlorophyll, photosynthetic efficiency [carbon dioxide (¹⁴CO₂)-fixation], relative water content (RWC) and abscisic acid (ABA) content. Pretreatment of seeds with SA alleviated the Cd negative effect on these parameters. A drop in the activities of carboxylating enzymes, phosphoenol pyruvate carboxylase (PEPC, EC 4.1.1.31), and ribulose1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) were observed in Cd treated plants. Pretreatment with SA ameliorated the inhibitory effect of Cd on the studied enzymatic activities. Cadmium applied to salicylic acid free plants increased the level of stress metabolites, malondialdehide (MDA), hydrogen peroxides, and free proline content. Salicylic acid pretreatment decreased the toxic effect level of cadmium manifested by lower lipid peroxidation, lesser production of H₂O₂ and reduction in free proline content. The ultrastructural investigation of the root cells of wheat exposed to different concentrations of Cd for 15 days was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis, highly condensed nuclear chromatin and increment of disintegrated organelles. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. An important issue arising from this study was how the short-term treatment with SA affected several physiological processes, such as plant growth, photosynthesis, and antioxidant defense system. We assume that the beneficial effect of SA during an earlier growth period could be related to avoidance of cumulative damage upon exposure to cadmium. One of the important roles of SA in inducing resistance to various environmental stresses is manifested by its ability to express genes that code for pathogenesis-related proteins or defense-related enzymes. Also, SA could form a complex with Cd that may provide Cd tolerance.