Morpho-physiological parameters affecting iron deficiency chlorosis in soybean (Glycine max L.)

Background and aims

Iron deficiency chlorosis (IDC) leads to severe leaf chlorosis, low photosynthetic rates, and yield reductions of several million metric tonnes each year. In order to devise breeding and genetic transformation programs that aim at generating high-yielding and IDC-tolerant soybean lines, it is necessary to better understand the mechanisms that enable tolerant plants to survive under Fe-limiting conditions.

Methods

An in silico analysis in the USDA soybean collection allowed the identification of a set of novel efficient and inefficient soybean cultivars which can be used in future studies concerning IDC response. Plants were grown in iron deficient and iron sufficient conditions using a bicarbonate system and several IDC-related aspects were studied.

Results

A new set of efficient and inefficient soybean lines were identified in silico, and their tolerance to IDC was confirmed under laboratorial conditions. New plant traits that are highly correlated to IDC scoring were identified: a negative correlation was found between SPAD values and stem weight, weight of the unifoliolates and iron concentration of the first unifoliolates was found; higher SPAD values were correlated with the amount of iron in the first trifoliate leaves. Our data also show that having higher concentrations of iron in the seeds provides increased resistance to IDC. No correlation was found between root iron reductase activity and chlorosis.

Conclusions

Soybean differential chlorosis susceptibility between different accessions is linked to specific morpho-physiological parameters such as unifoliolate leaf size, stem weigh, concentration of iron in the seeds, and tissue iron partitioning.     

Selection of olive varieties for tolerance to iron chlorosis

Under certain conditions, olive trees grown on calcareous soils suffer from iron chlorosis. In the present study several olive varieties and scion-rootstock combinations were evaluated for their tolerance to iron chlorosis. Plants were grown over several months in pots with a calcareous soil, under two fertilization treatments. These consisted of periodic applications of nutrient solutions containing either, 30 μmol/L FeEDDHA or not Fe. Tolerance was assessed by the chlorosis and growth parameters of plants grown without Fe, compared to those plants grown with Fe. Results show that there are differences in tolerance among olive varieties and that tolerance is mainly determined by the genotype of the rootstock. These results open the way to use tolerant varieties for those conditions where iron chlorosis could become a problem.     

Bicarbonate concentration as affected by soil water content controls iron nutrition of peanut plants in a calcareous soil.

Strategy I peanut plants are frequently subjected to iron deficiency when growing in calcareous soils, which contain high concentrations of bicarbonate. In calcareous soils under field conditions, it has been noted that chlorosis increases in severity after excessive rainfall or irrigation, but the chlorosis symptoms of peanuts are alleviated after waterlogged soils dry. A pot experiment was conducted simulating the chlorosis symptom observed in the field when peanut plants are exposed to fluctuating soil water content induced from rainfall or irrigation. We investigated the bicarbonate fluctuations resulting from adjustable soil water content (SWC) that could lead to bicarbonate-induced iron chlorosis of peanuts growing in calcareous soil. The experiments included three treatments of SWC (50% of water holding capacity (WHC), 80% of WHC, and 100% of WHC) under two levels of CaCO(3) concentrations (at 8.67% and 18.67%.) The results showed that the iron nutrition of peanuts could be regulated by different SWC at both CaCO(3) levels. Our observations indicate that iron deficiency chlorosis symptoms in peanuts grown in high soil water content were more severe, compared to those of peanuts in lower soil water content. A shift from high soil water content to lower soil water content could improve or eliminate the iron deficiency chlorosis symptom of peanuts. The HCO(3)(-) concentration in the peanut rhizosphere increased with increasing SWC and CaCO(3) content and it correlated with the level of soil water content. We suggest that variations in the soil water content could induce HCO(3)(-) concentration variation in the rhizosphere of peanuts. Consequently, the high HCO(3)(-) concentration, which is induced by a high water content in calcareous soil and a high CaCO(3) level, could inhibit the physiological response to iron deficiency of peanuts, resulting in iron deficiency chlorosis. The study indicates that a reasonable agricultural practice of irrigation and drainage should be considered to improve and prevent iron deficiency chlorosis of strategy I plants in calcareous soil.     

Differences in responses to iron deficiency among various cultivars of mungbean (Vigna radiata (L.) Wilczek)

Ten mungbean cultivars were evaluated for their resistance to iron deficiency in view of chlorosis symptoms, plant growth and seed yield under field conditions on a calcareous soil in Thailand. The KPS2 cultivar was highly susceptible; the KPS1, PSU1 and Pag-asa 1 cultivars were somewhat susceptible; the VC1163B cultivar was moderately tolerant; the CN36, CN60, UT1 and CNM-I cultivars were tolerant; and the CNM8509B cultivar was very tolerant to iron deficiency. Foliar application of a solution of 5 g L^-1 ferrous sulphate was effective in correcting chlorosis that was induced by iron deficiency, and it enhanced both the growth and the yield of susceptible cultivars. Compared with the susceptible cultivar KPS2, the tolerant cultivar UT1 had a greater ability to lower the pH of the nutrient solution in response to iron deficiency. The root-associated Fe³⁺-reduction activity of UT1 that had been grown in -Fe medium was similar to that of the plants grown in +Fe medium when the acidification of the medium occurred. Acidification of the medium in response to iron deficiency might contribute to the efficient solubilization of iron from calcareous soils, and it related more closely to the resistance to iron deficiency than Fe³⁺ reduction by roots in mungbean cultivars.     

Effect of salt on physiological responses of barley to iron deficiency.

Iron chlorosis is very common on alkaline soils such as calcareous ones, since iron availability is limited by high pH. Under these conditions of iron deficiency, graminaceous plant species induce special mechanisms for iron acquisition, involving enhanced release of iron chelators called phytosiderophores. On the other hand, it is known that most of salt soils have alkaline pH. So, plants growing on this kind of soils are often subjected simultaneously to salinity and iron deficiency. This work aimed at (i) studying the physiological responses of barley (Hordeum vulgare L.) to iron deficiency, and (ii) evaluating the effect of salt on the iron nutrition and the phytosiderophore release. For this purpose, seedlings of Hordeum vulgare L. were cultivated under controlled conditions, either in a complete nutrient solution with or without NaCl, or in an iron free nutrient solution containing or not NaCl. The plant morphological aspect, chlorophyll content of young leaves, iron status, biomass production, and phytosiderophore release by roots were assessed. Plants subjected to Fe deficiency exhibited a severe chlorosis, accompanied by a significant biomass reduction. These plants developed more lateral roots than the control with a highly stimulated phytosiderophore release. However, the latter was greatly diminished when iron deficiency was associated to salinity. A depressive effect of salt on iron acquisition in plants subjected only to salt stress which was also observed and further confirmed by the important decrease of efficiency in iron acquisition. These results suggest that salinity may reduce capacity of plants to acquire iron from alkaline soils by inhibiting phytosiderophore release.     

Mapping genetic loci for tolerance to lime-induced iron deficiency chlorosis in grapevine rootstocks (Vitis sp.)

Iron is essential to plants for chlorophyll formation as well as for the functioning of various iron-containing enzymes. Iron deficiency chlorosis is a wide-spread disorder of plants, in particular, of those growing on calcareous soils. Among the different ways to control iron deficiency problems for crops, plant material and especially rootstock breeding is a suitable and reliable method, especially for fruit trees and grapes. The aim of the experiment was to characterize the genetic basis of grapevine chlorosis tolerance under lime stress conditions. A segregating population of 138 F1 genotypes issued from an inter-specific cross between Vitis vinifera Cabernet Sauvignon (tolerant) × V. riparia Gloire de Montpellier (sensitive) was developed and phenotyped both as cuttings and as rootstock grafted with Cabernet Sauvignon scions in pots containing non-chlorosing and chlorosing soils. Tolerance was evaluated by chlorosis score, leaf chlorophyll content and growth parameters of the shoots and roots. The experiments were performed in 2001, 2003 and 2006. The plants analysed in 2006 were reassessed in 2007. The most significant findings of the trial were: (a) the soil properties strongly affect plant development, (b) there are differences in tolerance among segregating genotypes when grown as cuttings or as rootstocks on calcareous soil, (c) calcareous conditions induced chlorosis and revealed quantitative trait loci (QTLs) implicated in polygenic control of tolerance, (d) rootstock strongly contributes to lime-induced chlorosis response, and (e) a QTL with strong effect (from 10 to 25 % of the chlorotic symptom variance) was identified on chromosome 13. This QTL colocalized with a QTL for chlorophyll content (R ² = 22 %) and a major QTL for plant development that explains about 50 % of both aerial and root system biomass variation. These findings were supported by stable results among the different years of experiment. These results open new insights into the genetic control of chlorosis tolerance and could aid the development of iron chlorosis-tolerant rootstocks.     

Iron deficiency in lentil: Yield loss and geographic distribution in a germplasm collection

Iron deficiency symptoms are observed on some genotypes of lentil (Lens culinaris Medikus) grown in calcareous soil. A germplasm collection of 3512 accessions originating from 18 countries was characterized for iron deficiency in a Calcic Rhodoxeralf soil at ICARDA, Tel Hadya, Syria in the 1979/80 season. At 105 days after sowing, 592 accessions, representing 16.9% of the collection, showed chlorosis symptoms characteristic of iron (Fe) deficiency. The Fe deficiency was verified by foliar application of Fe-chelate. Germplasm from different countries showed differences in iron deficiency, with those accessions exhibiting symptoms of iron deficiency mostly originating from relatively warm climates such as India (37.5% accessions showing Fe deficiency) and Ethiopia (30%). Populations from those Mediterranean countries where lentil originated (Syria and Turkey) exhibited Fe-deficiency symptoms only at very low frequencies. Fe-deficiency induced chlorosis was positively correlated with cold susceptibility. Fe chlorosis was transient, the deficiency symptoms largely disappearing during reproductive growth at a time, coinciding with increases in soil temperature and daylength-conditions favorable for plant growth. In Indian germplasm, mild deficiency symptoms did not lead to reduced seed yield, but there was a major yield reduction of 47% in those accessions with the most severe symptoms. Straw yields was reduced commensurately with the severity of symptoms. ei]Section editor: B G Rolfe     

Genome-Wide Association Studies Identifies Seven Major Regions Responsible for Iron Deficiency Chlorosis in Soybean (Glycine max)

Iron deficiency chlorosis (IDC) is a yield limiting problem in soybean (Glycine max (L.) Merr) production regions with calcareous soils. Genome-wide association study (GWAS) was performed using a high density SNP map to discover significant markers, QTL and candidate genes associated with IDC trait variation. A stepwise regression model included eight markers after considering LD between markers, and identified seven major effect QTL on seven chromosomes. Twelve candidate genes known to be associated with iron metabolism mapped near these QTL supporting the polygenic nature of IDC. A non-synonymous substitution with the highest significance in a major QTL region suggests soybean orthologs of FRE1 on Gm03 is a major gene responsible for trait variation. NAS3, a gene that encodes the enzyme nicotianamine synthase which synthesizes the iron chelator nicotianamine also maps to the same QTL region. Disease resistant genes also map to the major QTL, supporting the hypothesis that pathogens compete with the plant for Fe and increase iron deficiency. The markers and the allelic combinations identified here can be further used for marker assisted selection.     

石灰性土壤上HCO3-诱导花生缺铁失绿机制

采用土壤-营养液结合的分根培养方法,研究了部分根系供应HCO- 3或铁对花生铁营养的调控及其作用机制。结果表明,对花生部分根系供应HCO- 3或铁可以调控花生的铁营养,仅供HCO- 3可以诱导缺铁,而只供铁能矫正失绿,同时供应HCO- 3和铁时则不引起失绿。在花生新生叶失绿和复绿的过程中,其中的活性铁含量和全铁含量也有相应的消长。当花生表现缺铁失绿症状时,地上各部分的全铁含量显著降低,而土中根的全铁含量不降低、质外体铁含量升高。在HCO- 3存在的条件下,不同部分根系的铁( )还原酶活性因其生长介质而不同,营养液中根系的铁( )还原酶活性降低而土中根的铁( )还原酶活性不受影响。当花生表现缺铁失绿症状时,土壤中HCO- 3含量升高,有效铁含量不高,p H值无变化。因此,本试验证实了石灰性土壤上的高HCO- 3含量,主要是降低了花生地上部的铁含量而引起失绿,而且花生缺铁失绿又导致土壤HCO- 3含量升高     

Evaluation of different iron compounds in chlorotic Italian lemon trees (Citrus lemon).

The severe deficiency of iron or ferric chlorosis is a serious problem of most citrus trees established in calcareous soils, as a result of the low availability of iron in these soils and the poor uptake and limited transport of this nutrient in trees. The objective of this study was to evaluate the response of chlorotic Italian lemon trees (Citrus lemon) to the application of iron compounds to roots and stems. On comparing the effects of aqueous solutions of ferric citrate, ferrous sulphate and FeEDDHA chelate, applied to 20% of the roots grown in soil and sand, of trees that were planted in pots containing calcareous soil, it was observed that the chelate fully corrected ferric chlorosis, while citrate and sulphate did not solve the problem. EDDHA induced the root uptake of iron as well as the movement of the nutrient up to the leaves. With the use of injections of ferric solutions into the secondary stem of adult trees, ferric citrate corrected chlorosis but ferrous sulphate did not. The citrate ion expanded the mobility of iron within the plant, from the injection points up to the leaves, whereas the sulphate ion did not sufficiently improve the movement of iron towards the leaf mesophyll.     

冬小麦植物铁载体分泌的杂种效应

缺铁是石灰性土壤常见的植物营养问题之一.禾本科植物种或基因型的植物铁载体分泌能力与耐缺铁有关,提高植物铁载体分泌能力是改良缺铁的土壤上植物铁aestivum L.) 3个杂交种及其4个亲本在缺铁营养液中植物铁载体的分泌及杂种的效应.植物铁载体的分泌率通过根分泌物对新形成的Fe(OH)3的活化能力进行测定, 在缺铁症出现时每隔2、3天测定1次.在缺铁条件下,所有基因型都分泌较多的植物铁载体,并且随缺铁症状的发展分泌量增加.杂交种具有对缺铁更敏感的反馈系统,在缺铁条件下,杂交种比亲本分泌铁载体的速度更快、量更高.通过分析杂交种和亲本的关系,认为可以通过对亲本分泌植物铁载体能力和配合力的选择,利用杂种优势来提高小麦铁的利用效率.     

Evaluation of early generations for iron chlorosis in relation to productivity in groundnut (Arachis hypogaea L.)

Five popular but iron-inefficient cultivars were crossed with three efficient genotypes and both parents and F₁s were evaluated for iron-efficiency in potted calcareous and noncalcareous soil. The iron-efficient genotypes were dark green or green in both noncalcareous and calcareous soils whereas inefficient types were light green to yellow in calcareous soil. The chlorophyll and active iron (Fe²⁺) concentration of leaves was less in iron-efficient genotypes compared to efficient types in calcareous soil and reduction of both the parameters from noncalcareous to calcareous soil was considerably high in iron-inefficient lines. There was significant correlation between visual scores, chlorophyll and active iron content. There were no differences among F₁s for iron chlorosis and they were all iron-inefficient. The frequency of iron-inefficient plants was higher than the efficient plants in all F₂ populations. But most of the productive plants came from iron-efficient segregants indicating strong association between iron-efficiency and productivity. Based on the results selection for iron-efficiency in early generations and extensive evaluation for productivity in advanced generations is suggested for developing varieties for cultivation in calcareous soils.     

Iron availability in plant tissues-iron chlorosis on calcareous soils

The article describes factors and processes which lead to Fe chlorosis (lime chlorosis) in plants grown on calcareous soils. Such soils may contain high HCO₃ ^- concentrations in their soil solution, they are characterized by a high pH, and they rather tend to accumulate nitrate than ammonium because due to the high pH level ammonium nitrogen is rapidly nitrified and/or even may escape in form of volatile NH₃. Hence in these soils plant roots may be exposed to high nitrate and high bicarbonate concentrations. Both anion species are involved in the induction of Fe chlorosis.Physiological processes involved in Fe chlorosis occur in the roots and in the leaves. Even on calcareous soils and even in plants with chlorosis the Fe concentration in the roots is several times higher than the Fe concentration in the leaves. This shows that the Fe availability in the soil is not the critical process leading to chlorosis but rather the Fe uptake from the root apoplast into the cytosol of root cells. This situation applies to dicots as well as to monocots. Iron transport across the plasmamembrane is initiated by Fe^III reduction brought about by a plasmalemma located Fe^III reductase. Its activity is pH dependent and at alkaline pH supposed to be much depressed. Bicarbonate present in the root apoplast will neutralize the protons pumped out of the cytosol and together with nitrate which is taken up by a H⁺/nitrate cotransport high pH levels are provided which hamper or even block the Fe^III reduction.Frequently chlorotic leaves have higher Fe concentrations than green ones which phenomenon shows that chlorosis on calcareous soils is not only related to Fe uptake by roots and Fe translocation from the roots to the upper plant parts but also dependent on the efficiency of Fe in the leaves. It is hypothesized that also in the leaves Fe^III reduction and Fe uptake from the apoplast into the cytosol is affected by nitrate and bicarbonate in an analogous way as this is the case in the roots. This assumption was confirmed by the highly significant negative correlation between the leaf apoplast pH and the degree of iron chlorosis measured as leaf chlorophyll concentration. Depressing leaf apoplast pH by simply spraying chlorotic leaves with an acid led to a regreening of the leaves.     

Functional Analysis of the Genotypic Differences in Response of Pea (<i>Pisum sativum</i> L.) to Calcareous-Induced Iron Deficiency

Lime-induced iron chlorosis is a major nutritional disorder causing severe plant growth and yield reduction in the calcareous soils of Tunisia. The understanding the behavior of key metabolic functions of peas on calcareous soils, the identification of useful traits of tolerance, and the exploration of the genotypic differences in response to this constraint remain the most efficient approaches due to their coast, environmental benefits, and sustainability. For this purpose, a greenhouse experiment was conducted on three pea genotypes (Alexandra: Alex, Douce de provence: DP, and Merveille de Kelvedon: MK) cultivated on calcareous soil (Fe-deficient) and fertile soil (control). Plant growth, SPAD index, iron nutrition and distribution, photosynthesis, and antioxidant enzymes were deeply analyzed to discriminate genotypic differences. Calcareous-induced iron deficiency reduced SPAD index, plant growth, net photosynthesis, and tissue Fe content against a significant stimulation of the oxidative stress indicators, H₂O₂ and Malondialdehyde (MDA). Moreover, we reported a significant induction of SOD and CAT activity in shoots and roots of the Alexandra genotype. Fe use efficiency increased on calcareous soil and clearly discriminated the studied genotypes. Alexandra genotype was found to be the most tolerant to lime-induced iron chlorosis. This genotype protects its tissues against oxidative stress by stimulating enzyme activities (SOD and CAT) and develops significant efficiency of Fe uptake, translocation to shoots and use when cultivated on calcareous soil.     

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.     

Behaviour of iron and manganese chelates in calcareous soils and their effectiveness for plants

Summary The behaviour of the metal chelates Mn-EDTA, Mn-DTPA, Mn-EDDHA, Fe-EDTA, Fe-DTPA and Fe-EDDHA in calcareous soils and their availability to plants were studied. The effectiveness of a metal chelate was shown to depend on its stability, the fixation capacity in the soil and its toxicity to the plant. Incorporation of Fe-DTPA into a framework of silica molecules prevents the fixation of Fe-DTPA in the soil. Fe-DTPA and Fe-EDDHA cause a reduction in the manganese uptake of the plant. The most striking result was the behaviour of Mn-DTPA in calcareous soils. Partial replacement of manganese in the chelate by iron from the soil makes this chelate useful for supplying the plant with both iron and manganese. Mn-DTPA appears to be the ideal type of chelate for the correction of chlorosis in the Netherlands, but unfortunately is not yet commercially available.     

Accumulation of apoplastic iron in plant roots : a factor in the resistance of soybeans to iron-deficiency induced chlorosis?

We hypothesized that the resistance of Hawkeye (HA) soybean (Glycine max L.) to iron-deficiency induced chlorosis (IDC) is correlated to an ability to accumulate a large pool of extracellular-root iron which can be mobilized to shoots as the plants become iron deficient. Iron in the root apoplast was assayed after efflux from the roots of intact plants in nutrient solution treated with sodium dithionite added under anaerobic conditions. Young seedlings of HA soybean accumulated a significantly larger amount of extracellular iron in their roots than did either IDC-susceptible PI-54619 (PI) soybean or IDC-resistant IS-8001 (IS) sunflower (Helianthus annus L.). Concurrently, HA soybean had much higher concentrations of iron in their shoots than either PI soybean or IS sunflower. The concentration of iron in the root apoplast and in shoots of HA soybean decreased sharply within days after the first measurements of extracellular root iron were made, in both +Fe and −Fe treatments. The accumulation of short-term iron reserves in the root apoplast and translocation of iron in large quantities to the shoot may be important characteristics of IDC resistance in soybeans.     

Iron deficiency induces cluster (proteoid) root formation in Casuarina glauca

The effect of iron deficiency, phosphorus, NaHCO₃, chelator supply and nitrogen source on the formation of cluster (proteoid) roots was investigated in Casuarina glauca growing in water culture. The addition of iron-binding chelators (e.g. EDDHA, DTPA, EDTA) or increase in nutrient solution pH with NaHCO₃ resulted in the formation of cluster roots when plants were grown in solution lacking iron. Phosphorus supply even at a concentration of 500 µM did not inhibit cluster root formation if EDDHA was added to the iron-deficient medium. Cluster root formation was influenced significantly by nitrogen source and occurred only in nitrate-fed plants.C. glauca seemed to be very sensitive to iron deficiency as shown by plant chlorosis when grown on alkaline soil. The symptoms of chlorosis decreased as the chlorophyll content in shoots and the number of cluster roots increased, suggesting that the alleviation of iron deficiency in plant tissues was correlated with cluster root formation. It appears that iron deficiency is more important than phosphorus deficiency in inducing the formation of cluster roots in C. glauca.