Root Ferric Chelate Reductase Activity Research Articles

Brief characterisation of Fe chlorosis in chia (Salvia hispanica L.) plants grown in nutrient solution

ABSTRACT Chia (Salvia hispanica L.) plant is a well-known plant due to the nutraceutical value of its seeds. The aim of this preliminary study was to assess the response of Chia plants to Fe deficiency. Chia plants were grown for 12 days in Hoagland’s nutrient solutions without Fe (Fe0) and with Fe (Fe10-10 µM Fe). Biomass parameters and root ferric chelate-reductase activity (FC-R; EC 1.16.1.17) were determined at the end of the experiment. Chlorophyll estimations (expressed as SPAD readings) decreased progressively, showing the typical symptoms of iron chlorosis. In addition, iron-deficient chia plants exhibit smaller biomass (number of leaves, root, and shoot growth reduction) compared to control plants. These plants also showed morphological changes in roots. Furthermore, root FC-R activity was significantly lower in Fe0 plants.

Silencing of FRO1 gene affects iron homeostasis and nutrient balance in tomato plants

AbstractBackgroundIron chlorosis is an abiotic stress of worldwide importance affecting several agronomic crops. It is important to understand how plants maintain nutrient homeostasis under Fe deficiency and recovery.AimsWe used the virus‐induced gene silencing (VIGS) method to elucidate the role of the FRO1 gene in tomato plants and identify the impact on regulation of the root ferric‐chelate reductase (FCR) activity and nutritional homeostasis.MethodsTomato plantlets cv. “Cherry” were transferred into half‐strength Hoagland's nutrient solution containing 0.5 µM of Fe (Fe0.5). In phase I, two treatments were established: control (Fe0.5) plants and VIGS‐0.5 plants corresponding to plants with the FRO1 gene silenced. In phase II, plants from Fe0.5 and VIGS‐0.5 were transferred to new nutrient solution and then grown for a further 14 days under 0 and 10 µM of Fe (as 0.5 µM would not be enough for the larger plants during phase II). Therefore, four treatments were imposed: Fe0, Fe10, VIGS‐0, and VIGS‐10.ResultsVIGS‐0.5 plants had significantly lower chlorophyll (Chl) and root FCR activity compared to the respective non‐silenced plants and retained more Cu and Zn in the roots at the expense of stems (Cu) or young leaves (Zn). Iron concentration in roots and stems decreased in FRO1 gene‐silenced plants, compared to control plants, but the allocation to different organs was similar in both treatments.ConclusionsThere was a partial recovery of leaf Chl in the VIGS‐10 plants and a higher concentration of Fe in all organs. In contrast, the allocation of Cu to roots decreased in the VIGS‐10 plants.

Apoplast utilisation of nanohaematite initiates parallel suppression of RIBA1 and FRO1&3 in Cucumis sativus

Nanoscale Fe containing particles can penetrate the root apoplast. Nevertheless, cell wall size exclusion questions that for Fe mobilisation, a close contact between the membrane integrating FERRIC REDUCTASE OXIDASE (FRO) enzymes and Fe containing particles is required. Haematite nanoparticle suspension, size of 10–20 nm, characterized by 57Fe Mössbauer spectroscopy, TEM, ICP and SAED was subjected to Fe utilisation by the flavin secreting model plant cucumber (Cucumis sativus). Alterations in the structure and distribution of the particles were revealed by 57Fe Mössbauer spectroscopy, HRTEM and EDS element mapping. Biological utilisation of Fe resulted in a suppression of Fe deficiency responses (expression of CsFRO 1, 2 &3 and RIBOFLAVIN A1; CsRIBA1 genes and root ferric chelate reductase activity). Haematite nanoparticles were stacked in the middle lamella of the apoplast. Fe mobilisation is evidenced by the reduction in the particle size. Fe release from nanoparticles does not require a contact with the plasma membrane. Parallel suppression in the CsFRO 1&3 and CsRIBA1 transcript amounts support that flavin biosynthesis is an inclusive Fe deficiency response involved in the reduction-based Fe utilisation of Cucumis sativus roots. CsFRO2 is suggested to play a role in the intracellular Fe homeostasis.

Analysis of contrast iron chlorosis tolerance in the pear cv. ‘Huangguan’ grafted onto pyrus betulifolia and quince A grown in calcareous soils

Quince (Cydonia oblonga Mill.) represents an examplar rootstock in modern training programmes to escalate pear production worldwide because of its dwarfing characteristic and precocity. However, iron deficiency often poses a hard hurdle to overcome for developing quince grafted pears in calcareous and alkaline soils. In North China, we observed that the high-quality pear cultivar Huangguan (Pynus bretschneideri Rehd.) grafted onto quince A (Hardy as interstock, defined as HG-QA) suffered iron deficiency chlorosis in calcareous soils in early spring, even more severe during late spring to early summer. In contrast, Huangguan grafted onto Pyrus betulifolia (Hardy as interstock, defined as HG-PB) did not suffer it. We were inspired to design this field experiment to figure out iron mobilization/uptake in the two rootstocks and the two scion-rootstock combinations. Our results showed that the soil plant analysis development (SPAD) values of quince A (24.7) and HG-QA (13.7) were much lower than that of Pyrus betulifolia (41.6) and HG-PB (36.1). The root length density and surface area density of quince A and HG-QA were more than 2-fold higher than those of Pyrus betulifolia and HG-PB. The rhizosphere pH of quince A and HG-QA was more than 0.20 units lower than that of Pyrus betulifolia and HG-PB respectively, whereas we didn’t detect any obvious differences in the root ferric-chelate reductase activity, iron concentrations in leaves and roots, and iron transport ratio. These results strongly suggest that the iron efficiency of HG-PB is attributed to an iron activation mechanism in leaves. The deciphering of the mechanisms that how rootstock influences the iron acquisition system inside the leaf of the scion and changes the efficiency of iron utilization will facilitate making effective solutions for iron deficiency induced chlorosis, popularizing and planting Huangguan grafted onto quince A in pear production.

Effects of iron and root zone pH on growth and physiological responses of paper birch (Betula papyrifera), trembling aspen (Populus tremuloides) and red-osier dogwood (Cornus stolonifera) seedlings in a split-root hydroponic system

Iron deficiency that is induced by high soil pH is a major factor affecting plant growth in calcareous soils and in some areas that have been reclaimed following industrial activities. Since the effects of high soil pH commonly involve Fe deficiency, in this study, we examined whether Fe provided to part of the root system exposed to low pH would alleviate high pH stress in paper birch (Betula papyrifera), trembling aspen (Populus tremuloides) and red-osier dogwood (Cornus stolonifera) seedlings. The plants were grown in a controlled environment growth room in mineral nutrient solution at pH 5 and 9 and provided with either 0 or 40 µM Fe in a split-root system for 8 weeks. At the end of the treatments, plant dry weights, net photosynthesis, transpiration rates, root ferric-chelate reductase activity, and leaf chlorophyll concentrations were measured, and elemental analyses were carried out in young leaves. The results demonstrated high root zone pH affected Fe, P and Zn concentrations in young leaves. In the three considered species, plants with part of their root system exposed to pH 5 had higher dry weights, net photosynthesis, and transpiration rates compared with the plants with the whole root system immersed in pH 9 solution. High root zone pH reduced photosynthesis, transpiration rates, leaf chlorophyll concentrations and the uptake of Fe, P, and Zn in plants. Partial exposure of the root system to low pH and Fe supply reduced leaf chlorosis and partly alleviated the high pH stress in the studied plants by improving Fe uptake, but did not alleviate root growth reductions.

Effects of different foliar iron applications on activity of ferric chelate reductase and concentration of iron in sweet potato (Ipomoea batatas)

We studied the relative efficacy of different forms of foliar iron (Fe) fertilisation on leaf re-greening in Fe-deficient, purple-fleshed sweet potato (Ipomoea batatas (L.) Lam.) varieties xuzi8 and xuzi6. Activities of ferric chelate reductase (FCR) and concentrations of Fe were measured in the leaves and roots at intervals over 5 days to quantify recovery from leaf chlorosis. Freshly expanded and chlorotic leaves were immersed in one of three different fertiliser compounds containing 9 mm Fe: FeSO4, Fe2(SO4)3, Fe(III)-EDTA. An Fe-sufficient treatment and an Fe-deficient control were included. The experiment had a completely randomised block design with five replications per treatment and was conducted in a sunlit glasshouse. For variety xuzi8, leaf FCR activity in the Fe2(SO4)3 treatment was highest at 1 h after application, and higher than all other treatments, whereas FeSO4 and Fe(III)-EDTA treatments showed their highest FCR at day 5 after application, both significantly higher than the Fe2(SO4)3 and control treatments. Furthermore, leaf Fe concentration reached a maximum in the FeSO4 treatment at day 1, and in the Fe2(SO4)3 treatment at day 3. By contrast, root Fe concentration was relatively constant and lower in the foliar Fe treatments than the Fe-sufficient and -deficient treatments. For variety xuzi6, leaf SPAD was higher with the Fe2(SO4)3 than the FeSO4 treatment at day 5 after application. In general, FCR activity and Fe concentrations in roots and leaves of xuzi6 were higher than those of xuzi8. Variations in leaf Fe concentrations were similar for both the FeSO4 and Fe2(SO4)3 treatments of the two varieties. Maximum leaf Fe levels in xuzi6 were ~4-fold those in xuzi8. The results of the study suggest that foliar-applied Fe2(SO4)3 was the most effective compound at correcting Fe-deficiency symptoms. The higher leaf and root FCR activity and Fe concentration in xuzi6 might explain its higher tolerance to Fe deficiency and better re-greening than xuzi8.

Plant growth promoting rhizobacteria enhanced leaf organic acids, FC-R activity and Fe nutrition of apple under lime soil conditions

Iron chlorosis in the calcareous soils is one most important stress factors worldwide that limits photosynthesis and decreases fruit yield and quality. Certain soil rhizobacteria produce organic compounds such as plant acids and they may reduce the soil rhizosphere pH and affect ferric chelate reductase (FC-R) activity in root. However, there is no knowledge regarding changes in organic acids content and FC-R activities of leaf due to rhizobacterial root inoculation. Therefore, the efficiency of six plant growth promoting rhizobacteria (PGPR) were tested on apple cv. Braeburn on M9 and MM106 rootstocks. The results of the experiment showed leaf organic acid contents, iron quantity of soil, root and leaf and root and leaf FC-R activity were significantly affected via rhizobacteria applications in apple plants. In MM106 and M9, there was a remarkable increase in Fe in M3 inoculated soil by 95 and 89%, respectively, compared to control. Average increases in citric, malic, malonic, butyric and lactic acid in the leaf were obtained from rhizobacterial root inoculations of 25.1, 21.8, 29.6, 18.0 and 18.2% in Braeburn/MM106, respectively. In Braeburn/M9, MFDCa1 application increased all organic acid concentrations compared to the control. MFDCa2 treatment caused the maximum leaf FC-R activity in Braeburn on M9 and MM106 (60.9 and 50.3 nmol Fe+2 g−1 FW h−1, respectively) while the least values were determined in the control (33.5 and 29.9 nmol Fe+2 g−1 FW h−1, respectively). This study showed the bacterial strains tested in our study may be used as a biofertilizer instead of Fe fertilizers.

Root plant growth promoting rhizobacteria inoculations increase ferric chelate reductase (FC-R) activity and Fe nutrition in pear under calcareous soil conditions

Iron deficiency occurring in calcareous soil is a problem in various plants. It is well known that some soil bacteria can release organic acids that can decrease the pH of the soil rhizosphere. However, there have been no attempts to study the effects of plant growth promoting rhizobacteria (PGPR), including organic acid releasing bacteria, on the organic acid contents of the leaf and FC-R activity in the roots and leaves under calcareous soil conditions. Therefore, pear plants were inoculated with 6 bacterial strains with the aim of acquiring iron under calcareous conditions. Uniform 1-year-old pear cv. Deveci sapling grafted on BA-29 and OHF-333 rootstocks were planted in plastic pots containing 10L of loamy soil at 29.6% CaCO3. All bacteria were applied to the roots as an inoculation before planting. The root and leaf Fe content, FC-R activity, leaf organic acids, and soil Fe content were compared in the Alcaligenes 637Ca, Agrobacterium A18, Staphylococcus MFDCa1, MFDCa2, Bacillus M3 and Pantoea FF1 strains. The study showed that the leaf organic acid content and the Fe content in the soil, root and leaf were significantly affected by the bacterial treatments in pear plants. It was determined that the total and active Fe in the leaf was higher in OHF-333 compared to BA-29 by 7% and 14%, respectively. Furthermore, the leaf FC-R activity of Deveci on OHF-333 was 8% higher than that on BA-29. In the Deveci/BA-29 plants, the 637Ca treatment had the highest root FC-R activity value (107nmolFe+2gr−1FW h−2). The highest leaf FC-R activity value was obtained from the MFDCa1, MFDCa2 and FF1 treatments (58.4, 56.3 and 55.7nmolFe+2gr−1FWh−2, respectively). The bacterial strains used in the present study have an important potential to be used as a biofertilizer to replace the use of iron fertilizers.

Nitric oxide signaling is involved in the response to iron deficiency in the woody plant Malus xiaojinensis

To cope with iron (Fe) deficiency, plants have evolved a wide range of adaptive responses from changes in morphology to altered physiological responses. Recent studies have demonstrated that nitric oxide (NO) is involved in the Fe-deficiency response through hormonal signaling pathways. Here, we report that NO plays a significant role in Malus xiaojinensis, an Fe-efficient woody plant. Fe deficiency triggered significant accumulation of NO in the root system, predominantly in the outer cortical and epidermal cells of the elongation zone. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) completely arrested Fe deficiency-induced root hair formation, blocked the increase in root ferric-chelate reductase activity and in root H+ excretion, further reduced the active iron content in young leaves and roots, and prevented the upregulation of the critical Fe-related genes, FIT, MxFRO2-like, and MxIRT1. These conditions were restored under Fe deficiency by treatment with the NO donor, sodium nitroprusside (SNP). Additionally, chlorophyll content and relative expression levels of the genes chlorophyll a deoxygenase (MxCAO) and polyamine oxidase (MxPAO) were not changed significantly following Fe deficiency for 6 d; however, SNP treatment increased MxHEMA gene expression. Interestingly, the Fv/Fm ratio, the maximum quantum yield of photosystem II (PSII), decreased significantly following cPTIO treatment. We observed more severe chlorosis under Fe deficiency with cPTIO treatment for 9 d. These results strongly suggest that NO mediates a range of responses to Fe deficiency in M. xiaojinensis, from morphological changes to the regulation of physiological processes and gene expression.

Ferric-chelate reductase activity is a limiting factor in iron uptake in spinach and kale roots

Iron (Fe) is essential for many vital processes in plants, including chlorophyll biosynthesis, DNA synthesis, nitrogen reduction, and photosynthetic electron transfer. However, free Fe ions also participate in the Fenton reaction, which generates reactive oxygen species that induce oxidative stress, leading to lipid peroxidation, protein oxidation, and DNA mutation. Accordingly, plants have developed strategies to prevent roots from absorbing excess Fe. Here, we investigated Fe homeostasis in hydroponically grown spinach and kale under various Fe concentrations (5-800 µM), as well as the resulting changes in bioactive compound levels. Spinach and kale Fe contents did not increase under Fe concentrations in the nutrient solution of up to 200 µM. When spinach plants (grown in nutrient solution containing 2 µM Fe-EDTA) were transferred to 50 µM Fe-EDTA, ferric-chelate reductase activity in roots rapidly decreased within 24 hours. In kale, ferric-chelate reductase activity also significantly decreased with increasing Fe-EDTA concentration. As Fe did not accumulate in spinach plants, no significant differences in plant growth were observed. However, in kale, root growth was slightly suppressed by high Fe (100-200 µM) levels in the rhizosphere. Comparisons to other studies suggest that variations in phenolic and flavonoid contents are dependent on plant species, but overall, Fe treatment did not result in a significant increase in these compounds in spinach or kale. Our results suggest that in spinach and kale roots, the regulation of Fe contents by ferric-chelate reductase is responsible for maintaining Fe homeostasis.

Alkaline stress and iron deficiency regulate iron uptake and riboflavin synthesis gene expression differently in root and leaf tissue: implications for iron deficiency chlorosis.

Iron (Fe) is an essential mineral that has low solubility in alkaline soils, where its deficiency results in chlorosis. Whether low Fe supply and alkaline pH stress are equivalent is unclear, as they have not been treated as separate variables in molecular physiological studies. Additionally, molecular responses to these stresses have not been studied in leaf and root tissues simultaneously. We tested how plants with the Strategy I Fe uptake system respond to Fe deficiency at mildly acidic and alkaline pH by measuring root ferric chelate reductase (FCR) activity and expression of selected Fe uptake genes and riboflavin synthesis genes. Alkaline pH increased cucumber (Cucumis sativus L.) root FCR activity at full Fe supply, but alkaline stress abolished FCR response to low Fe supply. Alkaline pH or low Fe supply resulted in increased expression of Fe uptake genes, but riboflavin synthesis genes responded to Fe deficiency but not alkalinity. Iron deficiency increased expression of some common genes in roots and leaves, but alkaline stress blocked up-regulation of these genes in Fe-deficient leaves. In roots of the melon (Cucumis melo L.) fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These results suggest a model implicating shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots.

The memory of iron stress in strawberry plants

To provide information towards optimization of strategies to treat Fe deficiency, experiments were conducted to study the responses of Fe-deficient plants to the resupply of Fe. Strawberry (Fragaria × ananassa Duch.) was used as model plant. Bare-root transplants of strawberry (cv. ‘Diamante’) were grown for 42 days in Hoagland's nutrient solutions without Fe (Fe0) and containing 10 μM of Fe as Fe-EDDHA (control, Fe10). For plants under Fe0 the total chlorophyll concentration of young leaves decreased progressively on time, showing the typical symptoms of iron chlorosis. After 35 days the Fe concentration was 6% of that observed for plants growing under Fe10. Half of plants growing under Fe0 were then Fe-resupplied by adding 10 μM of Fe to the Fe0 nutrient solution (FeR). Full Chlorophyll recovery of young leaves took place within 12 days. Root ferric chelate-reductase activity (FCR) and succinic and citric acid concentrations increased in FeR plants. Fe partition revealed that FeR plants expressively accumulated this nutrient in the crown and flowers. This observation can be due to a passive deactivation mechanism of the FCR activity, associated with continuous synthesis of succinic and citric acids at root level, and consequent greater uptake of Fe.

EFFECT OF IRON DEFICIENCY ON ROOT FERRIC CHELATE REDUCTASE, PROTON EXTRUSION, BIOMASS PRODUCTION AND MINERAL ABSORPTION OF CITRUS ROOT STOCK ORANGE JASMINE (MURRAYA EXOTICA L.)

Three-week iron (Fe) deficiency stress experiments were conducted using two citrus root stocks, Fe-deficiency tolerant Orange Jasmine (OJ, Murraya exotica L.) and the sensitive Flying Dragon [FD, Poncirus trifoliata var. monstrosa (T. Ito) Swingle]. Root ferric chelate reductase activity and proton extrusion increased in OJ between 12 and 18 d of stress, whereas there was no change in FD. Dry weight of OJ roots increased in contrast to FD which decreased. The Mn content in OJ remained the same even under Fe stress. Zn content in OJ roots doubled while that of FD increased 4-folds. The shoot/root Fe accumulation ratio increased in OJ while it decreased in FD. OJ apparently has mechanisms for increasing root biomass, controlling Fe reutilization and regulating manganese (Mn) and zinc (Zn) absorption in response to Fe deficiency. These mechanisms could help maintain homeostasis under heavy metal stress, which would be useful for improved growth of economically important citrus species.

RELATIVE SUSCEPTIBILITY OF SOME PRUNUS ROOTSTOCKS IN HYDROPONICS TO IRON DEFICIENCY

The susceptibility of eleven Prunus rootstocks to iron (Fe) deficiency was studied in hydroponics by growing them with 20 μM Fe, 0 μM Fe or 3 μM Fe+10 mM sodium bicarbonate. Based on the intensity of leaf chlorosis, the peach-almonds PR 204/84, Stylianidis K and KID2, produced at the Pomology Institute of Naoussa (Greece), showed the same or even greater tolerance than GF 677, the Greek peach-almond Retsou x Nemaguard, the plum-almond Myrandier 617 and the peaches GF 305, IDS 37, Greek wild peach seedling the greatest susceptibility whereas the plums St. Julien GF655/2 and Myrobalan 29C intermediate. Rootstocks without Fe presented significantly lower nitrogen and Fe whereas with bicarbonate significantly lower nitrogen, phosphorus, Fe and zinc. Root ferric chelate reductase activity was significantly increased in −Fe rootstocks but negatively correlated with their tolerance; physiological and morphological changes were observed along a zone of a few centimeters length, 1–2 mm behind the root tip.

Nitric Oxide Acts Downstream of Auxin to Trigger Root Ferric-Chelate Reductase Activity in Response to Iron Deficiency in Arabidopsis

In response to iron (Fe) deficiency, dicots employ a reduction-based mechanism by inducing ferric-chelate reductase (FCR) at the root plasma membrane to enhance Fe uptake. However, the signal pathway leading to FCR induction is still unclear. Here, we found that the Fe-deficiency-induced increase of auxin and nitric oxide (NO) levels in wild-type Arabidopsis (Arabidopsis thaliana) was accompanied by up-regulation of root FCR activity and the expression of the basic helix-loop-helix transcription factor (FIT) and the ferric reduction oxidase 2 (FRO2) genes. This was further stimulated by application of exogenous auxin (α-naphthaleneacetic acid) or NO donor (S-nitrosoglutathione [GSNO]), but suppressed by either polar auxin transport inhibition with 1-naphthylphthalamic acid or NO scavenging with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, tungstate, or N(ω)-nitro-L-arginine methyl ester hydrochloride. On the other hand, the root FCR activity, NO level, and gene expression of FIT and FRO2 were higher in auxin-overproducing mutant yucca under Fe deficiency, which were sharply restrained by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide treatment. The opposite response was observed in a basipetal auxin transport impaired mutant aux1-7, which was slightly rescued by exogenous GSNO application. Furthermore, Fe deficiency or α-naphthaleneacetic acid application failed to induce Fe-deficiency responses in noa1 and nial nia2, two mutants with reduced NO synthesis, but root FCR activities in both mutants could be significantly elevated by GSNO. The inability to induce NO burst and FCR activity was further verified in a double mutant yucca noa1 with elevated auxin production and reduced NO accumulation. Therefore, we presented a novel signaling pathway where NO acts downstream of auxin to activate root FCR activity under Fe deficiency in Arabidopsis.

Iron Deficiency‐induced Increase of Root Branching Contributes to the Enhanced Root Ferric Chelate Reductase Activity

In various plant species, Fe deficiency increases lateral root branching. However, whether this morphological alteration contributes to the Fe deficiency-induced physiological responses still remains to be demonstrated. In the present research, we demonstrated that the lateral root development of red clover (Trifolium pretense L.) was significantly enhanced by Fe deficient treatment, and the total lateral root number correlated well with the Fe deficiency-induced ferric chelate reductase (FCR) activity. By analyzing the results from Dasgan et al. (2002), we also found that although the two tomato genotypes line227/1 (P1) and Roza (P2) and their reciprocal F1 hybrid lines ("P1 x P2" and "P2 x P1") were cultured under two different lower Fe conditions (10(-6) and 10(-7) M FeEDDHA), their FCR activities are significantly correlated with the lateral root number. More interestingly, the -Fe chlorosis tolerant ability of these four tomato lines displays similar trends with the lateral root density. Taking these results together, it was proposed that the Fe deficiency-induced increases of the lateral root should play an important role in resistance to Fe deficiency, which may act as harnesses of a useful trait for the selection and breeding of more Fe-efficient crops among the genotypes that have evolved a Fe deficiency-induced Fe uptake system.

Iron Assimilation and Carbon Metabolism in ‘Concord’ Grapevines Grown at Different pHs

‘Concord’ grapevines (Vitis labruscana Bailey) are susceptible to lime-induced chlorosis, which decreases growth and productivity. In two separate experiments, we grew own-rooted vines in a peat–perlite medium adjusted to different pHs with CaCO3 to characterize how lime-induced Fe deficiency affects root and leaf ferric chelate reductase (FCR) and key enzymes and metabolites involved with glycolysis and the tricarboxylic acid (TCA) cycle in leaves. In addition, we measured the pH of the xylem sap as well as Fe, citrate, and malate concentrations. For both experiments, foliar levels of total Fe, active Fe (extracted in 0.1N HCl), and chlorophyll decreased as lime rate increased. An increase in root-medium pH from 5.8 to 7.5 resulted in a 10-fold increase in root FCR activity, whereas leaf FCR activity decreased 10-fold. An increase in root-medium pH did not raise xylem sap pH but decreased Fe and citrate to some extent. Xylem malate was highest at pH 6.6 and decreased both above and below this pH. Foliar data were evaluated in relation to active Fe content, because it is a better indicator of Fe nutritional status. Lower active Fe decreased midday CO2 assimilation and PSII quantum efficiency as well as night respiration. As active Fe decreased, aconitase activity decreased linearly, whereas the activity of glucose-6-phosphate dehydrogenase, NAD(P)-isocitrate dehydrogenase, NAD(P)-malic enzyme, malate dehydrogenase, phosphoenolpyruvate (PEP) carboxylase, PEP phosphatase, and pyruvate kinase increased curvilinearly. Glucose-6-phosphate, fructose-6-phosphate, and 3-phosphoglycerate content decreased curvilinearly as active Fe decreased. Malate content increased as active Fe increased to 1.0 mg·m−2 and then decreased above this level. Citrate increased linearly as active Fe decreased and was an order of magnitude lower than malate content. Our results suggest that leaf FCR activity may limit Fe assimilation to a greater extent than root FCR activity. The decreased leaf aconitase activity under Fe deficiency is the most likely cause of the increase in citrate levels. Greater activity of the other glycolytic and TCA enzymes under Fe deficiency may help to funnel carbon into the mitochondria and enhance NAD(P) reduction. Citrate levels (and the citrate:malate ratios) in the xylem exudate and leaf were much lower when compared with other species and may be linked to Fe inefficiency of ‘Concord’.

Iron Deficiency-Induced Secretion of Phenolics Facilitates the Reutilization of Root Apoplastic Iron in Red Clover

Phenolic compounds are frequently reported to be the main components of root exudates in response to iron (Fe) deficiency in Strategy I plants, but relatively little is known about their function. Here, we show that removal of secreted phenolics from the root-bathing solution almost completely inhibited the reutilization of apoplastic Fe in roots of red clover (Trifolium pratense). This resulted in much lower levels of shoot Fe and significantly higher root Fe compared with control and also resulted in leaf chlorosis, suggesting this approach stimulated Fe deficiency. This was supported by the observation that phenolic removal significantly enhanced root ferric chelate reductase activity, which is normally induced by plant Fe deficiency. Furthermore, root proton extrusion, which also is normally increased during Fe deficiency, was found to be higher in plants exposed to the phenolic removal treatment too. These results indicate that Fe deficiency-induced phenolics secretion plays an important role in the reutilization of root apoplastic Fe, and this reutilization is not mediated by proton extrusion or the root ferric chelate reductase. In vitro studies with extracted root cell walls further demonstrate that excreted phenolics efficiently desorbed a significant amount of Fe from cell walls, indicating a direct involvement of phenolics in Fe remobilization. All of these results constitute the first direct experimental evidence, to our knowledge, that Fe deficiency-induced secretion of phenolics by the roots of a dicot species improves plant Fe nutrition by enhancing reutilization of apoplastic Fe, thereby improving Fe nutrition in the shoot.

Uptake and Assimilation of Nitrate and Iron in Two Vaccinium Species as Affected by External Nitrate Concentration

Most Vaccinium species have narrow soil adaptation and are limited to soils that have low pH, high available iron (Fe), and nitrogen (N) primarily in the ammonium (NH4+) form. Vaccinium arboreum Marsh. is a wild species that can tolerate a wider range of soil conditions, including higher pH and nitrate (NO3-) as the predominant N form. This wider soil adaptation may be related to the ability of V. arboreum to acquire Fe and NO3- more efficiently than cultivated Vaccinium species, such as V. corymbosum L. interspecific hybrid (southern highbush). Nitrate and Fe uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in these two species grown hydroponically in either 1.0 or 5.0 mm NO3-. Nitrate uptake rate (on a whole-plant and FW basis) and root NR activity were significantly greater in V. arboreum compared with V. corymbosum. Iron uptake on a FW basis was also greater in V. arboreum, and was correlated with higher root FCR activity than was found in V. corymbosum. Increased Fe and NO3- uptake/assimilation in V. arboreum were reflected in increased organ and whole-plant dry weights compared with V. corymbosum. Vaccinium arboreum appears to be more efficient in acquiring and assimilating NO3- and Fe than is the cultivated species, V. corymbosum. This may partially explain the wider soil adaptation of V. arboreum.

Effect of External Nitrate Concentration on Nitrate and Iron Uptake and Assimilation in Vaccinium Species

Most Vaccinium species, including V. corymbosum, have strict soil requirements for optimal growth, requiring low pH, high iron, and nitrogen, primarily in the ammonium form. V. arboreum is a wild species adapted to high pH, low iron, nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake/assimilation compared with cultivated Vaccinium species. To test this, nitrate and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species, V. arboreum and the cultivated V. corymbosum. Plants were grown hydroponically for 15 weeks in either 1.0 or 5.0 mm NO3 with 0.09 mm Fe. Root FCR activity was greater in V. arboreum compared with V. corymbosum, especially at the lower external nitrate concentration. However, this was not reflected in differences in iron uptake. Nitrate uptake and root NR activity were greater in V. arboreum compared with V. corymbosum. The lower nitrate uptake and assimilation in V. corymbosum was reflected in decreased plant dry weight compared with V. arboreum. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity, and this may partially explain the wider soil adaptation of V. arboreum.