Nicotianamine and the distribution of iron into the apoplasm and symplasm of tomato (Lycopersicon esculentum Mill.)

The apoplasmic and symplasmic iron pools were determined in roots and leaves of Lycopersicon esculentum Mill. cv. Bonner Beste and its mutant chloronerva. The mutant is auxotrophic for the ubiquitous plant constituent nicotianamine (NA) and exhibits an impaired iron metabolism. Formation of apoplasmic iron pools in roots was dependent on the iron source in the nutrient solution. With Fe-ethylenediaminedi-(2-hydroxyphenylacetate) (FeEDDHA) only a very small apoplasmic iron pool was formed in the roots of both genotypes. Plants grown with FeEDTA increased their apoplasmic iron pool with increasing exogenous iron concentrations in the nutrient solution. The size of the apoplasmic pools in roots did not differ between the wild-type and the mutant (about 85 mol Fe · g^–1 DW). By contrast, the symplasmic iron concentrations in roots and leaves of the mutant were significantly higher when compared to the wild-type. An exogenous NA supply to the leaves of the mutant reduced the high symplasmic iron concentrations to the level of the wild-type. Mutant leaves exhibited a gradient of symplasmic iron concentrations depending on the developmental age of the leaves. The oldest leaves contained considerably more symplasmic iron than the youngest. The results demonstrate that the apparent iron deficiency of the mutant is not the consequence of an impaired iron transport from the apoplasm to the symplasm. Therefore, it is concluded that NA is not required for the transport of Fe(II) through the plasmalemma into the cell.Abbreviations BPDS bathophenanthroline disulfonic acid, Na₂ salt - FeEDDHA ferric N-N-ethylenediaminedi-(2-hydroxy-phenylacetate) - NA nicotianamine Part 40 in the series The normalizing factor for the tomato mutant chloronerva. For part 39 see Pich et al. (1991)The valuable technical assistance of Mrs. Christa Kallas and Mr. Günter Faupel is gratefully acknowledged.     

NicotJanamine and the Distribution of Iron into Apoplast and Symplast of Tomato (Lycopersicon esculentum Mill.): II. UPTAKE OF IRON BY PROTOPLASTS FROM THE VARIETY BONNER BESTE AND ITS NICOTIANAMINE-LESS MUTANT CHLORONERVA AND THE COMPARTMENTATION OF IRON IN LEAVES1

The influence of nicotianamine (NA) on the distribution of ironinto apoplast and symplast of a NA-containing tomato wild-typeand its NA-less mutant was investigated. Isolated protoplastsfrom wild-type and mutant leaves are able to reduce exogenousiron(III)citrate at equal rates. In spite of this, protoplastsfrom mutant leaves take up more iron from iron(III)citrate thanwildtype protoplasts. The mutant leaves accumulate higher amountsof iron in apoplast and symplast than wild-type leaves withan iron supply of 10 µM FeEDTA in nutrient solution. NAtreatment of the mutant leaves decreases both apoplasmic andsymplasmic iron in the direction of wild-type values. It isconcluded that NA is not essential for iron transport throughthe plasmalemma of protoplasts, but that endogenous NA decreasesthe high amount of iron in protoplasts by affecting the feed-backregulation of iron uptake by leaf cells.     

Phloem loading and unloading of sugars and amino acids

In terrestrial higher plants, phloem transport delivers most nutrients required for growth and storage processes. Some 90% of plant biomass, transported as sugars and amino nitrogen (N) compounds in a bulk flow of solution, is propelled though the phloem by osmotically generated hydrostatic pressure differences between source (net nutrient export) and sink (net nutrient import) ends of phloem paths. Source loading and sink unloading of sugars, amino N compounds and potassium largely account for phloem sap osmotic concentrations and hence pressure differences. A symplasmic component is characteristic of most loading and unloading pathways which, in some circumstances, may be interrupted by an apoplasmic step. Raffinose series sugars appear to be loaded symplasmically. However, sucrose, and probably certain amino acids, are loaded into minor veins from source leaf apoplasms by proton symporters localized to plasma membranes of their sieve element/companion cell (se/cc) complexes. Sucrose transporters, with complementary kinetic properties, are conceived to function as membrane transporter complexes that respond to alterations in source/sink balance. In contrast, symplasmic unloading is common for many sink types. Intervention of an apoplasmic step, distal from importing phloem, is reserved for special situations. Effluxers that release sucrose and amino acids to the surrounding apoplasm in phloem loading and unloading are yet to be cloned. The physiological behaviour of effluxers is consistent with facilitated membrane transport that can be energy coupled. Roles of sucrose and amino acid transporters in phloem unloading remain to be discovered along with mechanisms regulating symplasmic transport. The latter is hypothesized to exert significant control over phloem unloading and, in some circumstances, phloem loading.     

Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants

Background and Aims

Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues.

Methods

Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of ¹⁰⁹Cd applied at 34 nm was investigated between root and shoot.

Key Results

Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots.

Conclusions

Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots.     

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.     

In vivo quantification of cell coupling in plants with different phloem-loading strategies

Uptake of photoassimilates into the leaf phloem is the key step in carbon partitioning and phloem transport. Symplasmic and apoplasmic loading strategies have been defined in different plant taxa based on the abundance of plasmodesmata between mesophyll and phloem. For apoplasmic loading to occur, an absence of plasmodesmata is a sufficient but not a necessary criterion, as passage of molecules through plasmodesmata might well be blocked or restricted. Here, we present a noninvasive, whole-plant approach to test symplasmic coupling and quantify the intercellular flux of small molecules using photoactivation microscopy. Quantification of coupling between all cells along the prephloem pathways of the apoplasmic loader Vicia faba and Nicotiana tabacum showed, to our knowledge for the first time in vivo, that small solutes like sucrose can diffuse through plasmodesmata up to the phloem sieve element companion cell complex (SECCC). As expected, the SECCC was found to be symplasmically isolated for small solutes. In contrast, the prephloem pathway of the symplasmic loader Cucurbita maxima was found to be well coupled with the SECCC. Phloem loading in gymnosperms is not well understood, due to a profoundly different leaf anatomy and a scarcity of molecular data compared with angiosperms. A cell-coupling analysis for Pinus sylvestris showed high symplasmic coupling along the entire prephloem pathway, comprising at least seven cell border interfaces between mesophyll and sieve elements. Cell coupling together with measurements of leaf sap osmolality indicate a passive symplasmic loading type. Similarities and differences of this loading type with that of angiosperm trees are discussed.     

Influence of nicotianamine and iron supply on formation and elongation of adventitious roots in hypocotyl cuttings of the tomato mutant 'chloronerva' (Lycopersicon esculentum)

The influence of nicotianamine (NA) on formation and elongation of adventitious roots in hypocotyls of de-rooted NA-less mutant seedlings of Lycopersicon esculentum Mill, was examined in relation to the iron supply [ferric N-N'-ethylenediaminedi-(2-hydroxyphenylacetate) (FEDDHA), ferric ethylenediaminetetracetate (FeEDTA), ferric N-(2-hydroxyethyl)-ethylenediaminetriacetate (FeHEDTA, Fe-citrate and FeCl₃] in the nutrient solution. The initiation of root primordia in hypocotyl cuttings was independent of NA and occurred with about the same frequency in both, mutant and wild-type. In the mutant the development of primordia to adventitious roots was blocked at all iron sources used, except FeEDTA. Addition of NA (5x 10⁻⁶ to 2 × 10⁻⁵ M ) to the rooting medium resulted in a fast growth of adventitious roots in mutant cuttings with all iron sources tested. Rooting of wild-type cuttings was independent from NA application and iron sources. We suppose that NA is involved in the intracellular transport of iron. Its function is possibly linked with chelation of ferrous iron in the cell.     

Investigation of ascorbate-mediated iron release from ferric phytosiderophores in the presence of nicotianamine

Phytosiderophores (PS) are strong iron chelators, produced by graminaceous plants under iron deficiency. The ability of released PS to chelate iron(III), and subsequent uptake of this chelate into roots by YS1-type transport proteins, are well-known. The mechanism of iron release from the stable chelate inside the plant cell, however, is unclear. One possibility involves the reduction of ferric PS in the presence of an iron(II) chelator via ternary complex formation. Here, the conversion of ferric PS species by ascorbate in the presence of the intracellular ligand nicotianamine (NA) has been investigated at cytosolic pH (pH 7.3), leading to the formation of a ferrous NA chelate. This reaction takes place when supplying Fe(III) as a chelate with 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), with the reaction rate decreasing in this order. The progress of the conversion of ferric DMA to ferrous NA was monitored in real-time by high resolution mass spectrometry (FTICR-MS), and the results are complemented by electrochemical measurements (cyclic voltammetry), which allows detecting reactive intermediates and their change with time at high sensitivity. Hence, the combined results of electrochemistry and mass spectrometry indicate an ascorbate-mediated mechanism for the iron release from ferric PS, which highlights the role of ascorbate as a simple, but effective plant reductant.     

植物体内光合同化物韧皮部装载和卸出研究进展

近年来研究表明,植物体内光合同化物的韧皮部装载和卸出均有其本途径和质外体途径,装载转运的糖类主要有：（２）棉子糖及其人类似物（以共质体方式装载）;（２）蔗糖（以质外体方式装载）。同化物的共质体卸出可通过扩散和集中作用实现,而质外体卸出则根据蔗糖在质外体是否水解而分为两种类型。卸出和装载的途径、机理因植物种类及库源关系而不同,也会受生长发育阶段及环境的变化而调整。深入研究韧皮部装载和帛出调控机制,对     

湿地植物根表的铁锰氧化物膜

湿地植物根系具有泌氧能力 ,使其根表及根际微环境呈氧化状态。因而 ,土壤溶液中一些还原性物质被氧化 ,如 Fe2 ,Mn2 ,形成的氧化物呈红色或红棕色胶膜状包裹在根表 ,称为铁锰氧化物膜。铁锰氧化物膜及其根际微环境是湿地植物根系吸收养分和污染物的门户 ,势必会影响这些物质的吸收。主要综述了铁锰氧化物膜的形成和组成 ,以及根表形成的氧化物膜的生态效应 ,也就是氧化物胶膜对植物根系吸收外部介质中的养分及污染物质——重金属离子的影响     

Long-distance transport in non-vascular plants

Many macroalgae have significant spatial differentiation involving higher rate resource use at a site than of acquisition of that resource from the environment at that site. Long‐distance symplasmic transport of solutes occurs in some large green algae where the solutes are moved in streaming cytoplasm. In some large brown algae there is evidence of long‐distance symplasmic transport of organic C and other solutes. Structural and physiological data suggest that while the transport in ‘sieve tubes’ of Macrocystis might be by a Munch pressure flow mechanism the transport in many other brown algae is less likely to be by this mechanism. Less is known of long‐distance symplasmic transport in red algae. In terrestrial bryophytes transpiration occurs and in some liverworts and many mosses (but not in hornworts) there are files of dead cells in their tissues which may, and in some cases certainly, function in long‐distance apoplasmic water transport. The hydraulic conductivity of these conduits is poorly characterized. Long‐distance symplasmic transport in some mosses have been characterized both structurally and physiologically, but in other mosses and in liverworts the evidence is only structural. Most of these symplasmic transport pathways seem to have a high resistance to flow.     

Iron-containing particles accumulate in organelles and vacuoles of leaf and root cells in the nicotianamine-free tomato mutantchloronerva

Summary The mutantchloronerva ofLycopersicon esculentum Mill is the only known plant mutation that leads to a complete loss of the endogenous iron chelator nicotianamine. The mutant exhibits several morphological alterations and a permanent activation of the strategy I reactions of iron uptake as well as iron accumulation in roots and leaves. The electron microscopic energy loss technique of energy spectroscopic imaging (ESI) was used to localise the iron accumulated in the organs of wild-type and mutant plants. Iron-containing particles were detected in the chloroplast stroma and in vacuoles of mutant leaves, and in root cells in vacuoles and in mitochondria. In wild-type organs such particles were found at the same sites but they were smaller in size and occurred less frequently. The findings indicate that these compartments are preferential sites of iron storage or deposition in tomato tissues. It is discussed that the iron-containing particles detected are the result of iron release by oxidative stress. Application of nicotianamine to mutant plants, which reverts the mutant phenotype, led to a significant decrease of the iron-containing particles. This is seen as an indication that they may serve as intermediate iron stores and emphasises the crucial role of nicotianamine for the normal iron distribution in cells and organs.Dedicated Prof. Dr. K. Müntz on the occasion of his 65th birthday     

Phloem loading and transport characteristics of iron in interaction with plant-endogenous ligands in castor bean seedlings

The concentration of iron within cells has to be precisely regulated because shortage as well as surplus may be precarious for the survival of the cell. The maintenance of iron homeostasis in shoot organs requires an efficient signalling of the leaf cells’ iron status to the uptake sites of the roots. This ‘iron signal’ may be transferred by the phloem. The handling of iron in the symplast and during phloem transport calls for mechanisms taking into account the specific physicochemical properties of this element.Seedlings of Ricinus communis were used as model plants to investigate characteristic features of phloem loading, and of speciation and valence of iron during transport in the sieve tubes. When the storage endosperm is removed from the cotyledons, phloem is loaded from the reserve pool of the mesophyll cells. In this situation, iron and the other micronutrients copper, manganese and zinc are loaded in a constant stoichiometric ratio of 1:1 to the endogenous complexor nicotianamine (NA). Application of the chelators 1,4-di(4-phenylsulphonate)-1,10-phenanthroline (BPDS) and ethylenediaminetetraacetic acid to the cotyledon apoplast did not decrease the loading rate of iron, indicating symplastic loading. Supply of ferrous ions in various concentrations to the apoplast revealed the existence of two loading systems. One of them is linearly dependent upon the concentration, and remained unsaturated up to an apoplastic concentration of 200 μM. The other one, whose activity steeply inclines already with a slight increase of the apoplastic concentration, is saturable at a supply of 100 μM. The loading of iron is slowed down with time depending on whether iron is supplied to the apoplast as a complex with NA or citrate instead of as free ferrous ions. This effect may be caused by competition of these chelators with an iron uptake receptor in the plasmalemma of the mesophyll cells. In spite of the close relationship between iron and NA during phloem loading, the Fe-NA complex seems not to be the predominant transport species in the sieve tubes. A molecule of much larger mass than NA probably serves as a transport vehicle, as concluded from microdialysis experiments. Only 4% of the total iron in the sieve tube exudate was found to exist as Fe(II) and about 45% as Fe(III). The residue of more than 50% was tightly bound and not accessible even in the presence of the reductant sodium dithionite and the chelator BPDS. The conclusions regarding the nature of the transport species and the results on the valence of iron in the sieve tubes were confirmed by calculations with the software programs GEOCHEM [Sposito G, Mattigod SV (1979) A computer program for calculating chemical equilibria in soil solutions and other natural water systems. Kearney Foundation of Soil Sci, Univ. of California, Riverside, CA] and PHREEQC [Parkhurst (1995) PHREEQC-A computer program for speciation, reaction-path, adjective-transport, and inverse geochemical calculations. US Geological Survey]. A model is outlined on the basis of the experimental findings on the fate of iron from mobilisation in the endosperm to trans-chelation in the sieve tubes.     

Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

Iron reductase systems on the plant plasma membrane—A review

Higher plant roots, leaf mesophyll tissue, protoplasts as well as green algae are able to reduce extra-cellular ferricyanide and ferric chelates. In roots of dicotyledonous and nongraminaceous, monocotyledonous plants, the rate of ferric reduction is increased by iron deficiency. This reduction is an obligatory prerequisite for iron uptake and is mediated by redox systems localized on the plasma membrane. Plasma membrane-bound iron reductase systems catalyze the transmembrane electron transport from cytosolic reduced pyridine nucleotides to extracellular iron compounds. Natural and synthetic ferric complexes can act as electron acceptors.This paper gives an overview about the present knowledge on iron reductase systems at the plant plasma membrane with special emphasis on biochemical characteristics and localisation.     

SHORT COMMUNICATION:Response of nicotianamine synthase activity to Fe-deficiency in tobacco plants as compared with barley

In vitro nicotianamine synthase activity was measured in tobaccounder Fe-deficient or Fe-sufficient conditions. Its activitywas not induced by Fe-deficiency, in contrast to barley roots,implying that the molecular biological regulation of nicotianaminesynthase in response to Fe-deficiency may be different betweentobacco and barley. Key words: Barley, Fe-deficiency, ferric reduction, nicotianamine synthase, tobacco     

水稻土施硅对土壤-水稻系统中镉的降低效果

水稻中镉的积累造成人类健康的风险,增加水稻硅素能减轻镉中毒症状,降低稻米镉积累,但是硅对重金属的作用机理尚不清楚。主要研究了在中度和高度镉污染的土壤中,通过施用固态和液态的富硅物质对土壤-水稻系统中镉的吸收和转运的影响,探明决定镉和硅在根与芽的质外体和共质体中的作用机理。试验结果表明:(1)在中度和高度污染的土壤中,镉在土壤-作物系统中的转移和积累情况是不同的,可以通过富硅物质中的单硅酸与镉离子的相互作用,增加镉在硅物质表面的吸附来减少镉在土壤中的流动;(2)富硅物质可以降低水稻根和芽中镉的积累,在高度镉污染的情况下,施用硅可以使镉大量积累在水稻根及其共质体中,并降低根及其共质体中镉的转换和积累;(3)新鲜土壤中水萃取态的单硅酸含量与镉在土壤-作物系统中的流动性、转运以及积累等主要参数密切相关。     

Free radical involvement in the generation of trans‐root potential

The identity of the naturally occurring compounds that accept electrons from plasma membrane-bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical-generating and -quenching systems on the trans-root electrical potential, which had previously been shown to be coupled to plasma membrane-bound redox systems. The material was the primary root of 8-day-old maize (Zea mays L.) seedlings. Trans-root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans-root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans-root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane-bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.     

Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium.

Iron transport and utilization were examined in Bacillus megaterium Ard1, a mutant that is resistant to the hydroxymate antibiotic A22765 and whose growth is inhibited by the structurally similar hydroxamate Desferal. Rapid, low-level uptake of Desferal-50Fe was observed; such uptake was temperature and energy independent. Gel filtration chromatography of the cytoplasmic fraction of protoplasts labeled with Desferal-55Fe for 30 to 120 s demonstrated only unchanged esferal-55Fe in the cytoplasm. Although B. megaterium Ard1 showed transport of Desferal-59Fe by a process that resembles facilitated diffusion, this organism was unable to transfer iron from this chelate to cellular macromolecules for metabolic use. High-level transport of the ferric hydroxamate schizokinen-59Fe by B. megaterium Ard1 was both temperature and energy dependent. Within 30 s, protoplasts labeled with schizokinen-55Fe contained iron associated with certain macromolecules and in an apparent "pool" of schizokinen-55Fe in the cytoplasmic fraction. Prior transport of Dseferal-55Fe by protoplasts of strain Ard1 did not interfere with subsequent transport and utilization of schizokinen-59Fe. These studies suggest that transport of ferric hydroxamates may occur by a facilitated diffusion-type process; transfer of iron to cellular macromolecules may drive high-level transport of the chelate and may be the step at which energy is required in the iron transport-assimilation process.     

Energy requirements for maintenance of ion concentrations in roots

Maintenance of ion gradients across plant membranes is considered to be an important process requiring respiratory energy in plant tissues. In order to test this hypothesis, roots of intact plants of potato ( Solanum tuberosum L. cv. Alcmaria and cv. Pimpernel) were incubated in a closed circulation system. Electrical conductivity of the solution surrounding these roots was continuously monitored to determine total ion efflux into demineralized water. Anion efflux rate from the symplast was 35 neq (g dry weight)⁻¹ s⁻¹. In combination with literature data on the specific costs of ion transport, this efflux rate yields the respiration rate associated with re-uptake balancing efflux (i. e. maintenance of cellular ion concentrations). The results suggest that energy costs associated with re-uptake of ions may account for up to 25–50% of the total respiratory costs involved in ion influx.