De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

Effects of propylene and oxygen on the ethylene-producing system of apples

Hypobaric conditions and treatments with ethylene and the ethylene analogue propylene were used to investigate effects of oxygen and elhylene on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity and ethylene production of apples ( Malus sylveslris Mill. cv. Golden Delicious). Prcclimacteric apples were stored in air at 6.6 kPa (reduced pressure); 6.6 kPa ventilated with pure O₂; 6.6 kPa ventilated with 2600 μl 1⁻¹ C₂H₄; and in air at 101.3 kPa (atmospheric pressure) for 4 months at 4°C. No ACC synthase activity was detectable in apples stored at 6.6 kPa, whereas ACC synthase activity was induced in apples stored at 6.6 kPa and ventilated with either O₂ or C₂H₄. In a further experiment, preclimacteric apples were stored for 14 days either in air at 20 kPa or at 20 kPa ventilated with pure O₂. Both treatments were supplied with 58 500 μl 1⁻¹ propylene from day 0 to day 9 or from day 9 to day 12. Ethylene production of apples treated with propylene from day 0 to day 9 increased earlier than ethylene production of untreated apples. Propylene treatment from day 9 to day 12 did not stimulate ethylene production. Ethylene and propylene induced and stimulated extractable ACC synthase activity and ACC formation of apples. Oxygen enhanced this effect. The results also suggest inhibition of in vivo ACC synthase activity by propylene.     

Quantitative relationships between osmotic potential amd epicotyl growth in Vigna radiata as affected by osmotic stress and cotyledon excision

Ethylene biosynthesis in leaf discs of tobacco ( Nicotiana tabacum L. cv. Xanthi), as measured by the conversion of L-[3,4-¹⁴C]-methionine to ¹⁴C₂H₄, was markedly inhibited by exogenous ethylene. This inhibition was accompanied by a decrease in total (free + conjugated) content of 1-aminocyclopropane-1-carboxylic acid (ACC), most of which appeared in its conjugated inactive form. The autoinhibitory effect of ethylene was reversible and could be relieved by Ag⁺. The Ag⁺-treated leaf discs, with or without ethylene, contained only free ACC at an increased level. The results suggest that in tobacco leaves, the autoinhibition of ethylene production resulted from reduction in the availability of free ACC, through both suppression of ACC formation and increased ACC conjugation.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Stimulation of ACC-dependent ethylene production in citrus leaf discs by light

The influence of light and darkness incubation on in vivo ethylene forming enzyme (EFE) activity in citrus ( Citrus sinensis L. Osbeck cv. Salustiana) mature leaf discs was studied. Leaf discs incubated in light produced higher amounts of ethylene than in darkness. Transfer of discs from light to the dark resulted in a marked inhibition of EFE activity, whereas transfer of discs from the dark to light enhanced ethylene forming activity considerably. Light did not affect 1-aminocyclopropane-l-carboxylie acid (ACC) uptake. Incubation in a CO₂-eniiched atmosphere enhanced EFE activity both in light and in darkness, but light stimulation of EFE activity was apparently not affected by CO₂. Effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, inhibitor of photosynthetic electron flow) and KCN (inhibitor of cytochrome oxidase) were studied. DCMU at 0.2 m M inhibited EFE activity in light, whereas no effect was detected in the dark. On the other hand 1 m M KCN stimulated EFE activity in the light, and no significant effect was observed in the dark. CoCl₂ at 1 m M inhibited ACC-dependent ethylene production, suggesting that ethylene production from ACC is mediated by EFE in citrus leaf discs both in light and in the dark. Cycloheximide also inhibited EFE activity in the light and no effects were detected in the dark. Therefore protein synthesis in light (perhaps EFE synthesis) could be required for the light stimulation of the in vivo EFE activity.     

Partial purification and properties of a 36-kDa 1-aminocyclopropane-1-carboxylate N-malonyltransferase from mung bean

A 36-kDa 1-aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase, which converts the ethylene precursor ACC into the conjugated derivative malonyl-ACC (MACC), has been isolated from etiolated mung bean ( Vigna radiata ) hypocotyls, and partially purified in a four-step procedure. The enzyme is stimulated about 7-fold by 100 m M K⁺ salts or 0.5 m M Co²⁺ salts, and is inhibited competitively by D-phenylalanine (K_i= 1.3 m M ) and non competitively by CoASH (0.3 m M ). Beside malonyl-CoA, it is capable to use succinyl-CoA as an acyl donor. The 36-kDa enzyme described here exhibits a lower optimum temperature (40°C) and a 7- or 3-fold lower apparent K_m for ACC (68 μ M ) and malonyl-CoA (74 μ M ), respectively, when compared with its 55 kDa isoform already isolated from the same plant material. This data support the idea that several isoforms of ACC N-malonyltransferase exist in plants. These isoforms may play a differential role in regulating the availability of ACC, and consequently the rate of ethylene production, as well as detoxifying cells from D-amino acids.     

Inhibition of brassinosteroid-induced epinasty in tomato plants by aminooxyacetic acid and Co2+

The inhibitory effects of aminooxyacetic acid (AOA) and cobalt chloride (CoCl₂) on brassinosteroid (BR)-induced epinasty in tomato plants ( Lycopersicon esculentum Mill. cv. Heinz 1350) are evaluated. CoCl₂ dramatically decreases petiole bending and ethylene production as the concentration increases from 50 to 200 μ M. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petiole, instead of accumulating, is reduced and does not change over the concentration range tested. Inhibition of BR-induced epinasty by AOA results from inhibition of ACC synthesis. There are dramatic reductions in petiole bending, ethylene and ACC production as the concentration of AOA is increased from 50 to 200 μ M. Maximum inhibition occurs when the plants are pretreated with the inhibitors. The degree of inhibition increases as the length of pretreatment increases from 1 to 4 h. The response of BR-treated plants to AOA and CoCl₂ is similar to the effect of auxin, indicating the integral relationship between BR and auxin.     

Effect of short-chain fatty acids on the ethylene pathway in embryonic axes of Cicer arietinum during germination

The effect of short-chain saturated fatty acids (C₅–C₁₀) on the biosynthesis of ethylene in embryonic axes of chick-pea ( Cicer arietinum L.) seeds was investigated. The emergence of radicle and fresh weight of embryonic axes diminished with increasing number of carbons. The inhibition of germination caused by lower concentrations (1 m M ) of fatty acids (C₅–C₁₀) was partially reversed by exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), whereas exogenous ethylene was able to overcome the inhibitory effect provoked by all concentrations (1–5 m M ) of applied fatty acids (C₅–C₁₀). Ethylene production rates, and enzyme activities of ACC synthase and ACC oxidase decreased concomitantly with the molecular mass and increasing concentration of fatty acids. The inhibitory effect of these acids on ethylene production seems to result not only from a decreased ACC synthesis, but also from an enhancement of 1-malonylamino)cyclopropane-1-carboxylic acid (MACC) synthesis.     

Sulphate reduction in oxic and sub-oxic North-East Atlantic sediments

Abstract Oxic and sub-oxic N.-E. Atlantic sediments were examined for sulphate-reducing activity. Oxygen and/or nitrate reduction are probably the dominant mineralisation processes in the abyssal plain sediment studied. A low rate of sulphate reduction (0.1 nmol SO²⁻₄/ml/day) was recorded in the surface 5 cm of the continental slope sediment, together with the presence of a range of sulphate-reducing bacteria (SRB). A higher activity of sulphate reduction (2.2 nmol SO²⁻₄/ml/day) occurred in the continental shelf sediment which led to a small decrease in pore water sulphate and an increase in titration alkalinity. This sediment contained approx. 10²–10³ acetate, lactate and propionate oxidising SRB/ml. No low- M _r organic acids were detected in these sediments. However, amendment with 75 μM acetate stimulated sulphate-reducing activity in the shelf sediment.     

Ethylene synthesis and growth in embryogenic tissue of Norway spruce: Effects of oxygen, 1-aminocyclopropane-1-carboxylic acid, benzyladenine and 2,4-dichlorophenoxyacetic acid

Ethylene production from an embryogenic culture of Norway spruce ( Picea abies L.) was generally low. ca 2.5 nl g⁻¹ h⁻¹, whereas 1-aminocyclopropane-1 -carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g⁻¹ during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O₂) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 μ M ) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 μl I⁻¹) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2.4-dichloro-phenoxyacetic acid (2.4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g⁻¹ h⁻¹ within the 11-day incubation period. Although 2.4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g⁻¹, was observed in tissue treated with 2.4-D (22.5 μ M ) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent K_m was 50 μ M and V_max 2.7 nl g⁻¹ h⁻¹. Growth of the tissue was strongly inhibited by 2.4-D in the absence of BA, but weakly in the presence of BA (4.4 μ M ). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.     

Regulation by CO2 of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climacteric fruits

Cheverry, J. L., Sy, M. O., Pouliquen, J. and Marcellin, P. 1988. Regulation by CO₂ of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climateric fruits. - Physiol. Plant. 72: 535–540.
A high CO₂ concentration (20%) at 20°C rapidly and strongly inhibited the development of the climacteric ethylene burst in apple ( Malus domestica Borkh. cv. Granny Smith) and avocado ( Persea americana Mill. cv. Fuerte) fruits and did not change 1-aminocyclopropane-l-carboxylic acid (ACC) content. Treatment with 20% CO₂ markedly decreased ACC-dependent ethylene biosynthesis at 20°C in climacteric pericarp tissues. It is suggested, therefore, that high CO₂ levels inhibit conversion of ACC to ethylene.
Synthesis of the ethylene forming enzyme (EFE) was enhanced when intact preclimacteric apples or early climacteric avocados were pretreated for 40 h with 10 μ11^-1 ethylene. When CO₂ (20%) and ethylene were both applied, a reduced stimulatory effect of ethylene on EFE synthesis was observed. A high CO₂ concentration enhanced EFE acivity in excised tissues of apples and avocados incubated with ACC (2 m M ) and cycloheximide (1 m M ) or 2–5-norbornadiene (5 ml 1^-1). In the autocatalytic process, 20% CO₂ antagonized the stimulation of EFE synthesis by ethylene, but promoted EFE activity.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Involvement of abscisic acid and ethylene in the responses of citrus seedlings to salt shock

The responses of salt‐sensitive citrus rootstocks to 200 m M NaCl were periodically determined on seedlings of citrange Carrizo ( Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf) during 30 days. The stressed seedlings adjusted osmotically, reduced stomatal conductance, increased proline content and ethylene production, and showed massive leaf abscission (92%). The salt shock also increased abscisic acid (ABA) and aminocyclopropane‐1‐carboxylic acid (ACC) in roots, xylem fluid and leaves, and in addition promoted Cl⁻ accumulation. The pattern of change of ABA, ACC and proline followed a two‐phase response: an initial transient increase (10‐12 days) overlapping with a gradual and continuous accumulation. This biphasic response appears to be compatible with the proposal that the transitory hormonal rises are induced by the osmotic component of salinity, whereas the Cl⁻ increase determines the subsequent accumulations. During the second phase, Cl⁻ levels correlated with abscission in leaves. Production of leaf ethylene was also concomitant with the increase in the abscission rate. Salt‐induced abscission was either reduced with CoCl₂ (52%) or inhibited with silver thiosulphate (14%). The results suggest that in salt‐stressed citrus, leaf abscission is induced by the chloride build‐up through a mechanism that stimulates leaf ACC synthesis and further conversion to ethylene.     

Brassinosteroid-induced exaggerated growth in hydroponically grown Arabidopsis plants

The effects of root application of brassinolide (BL) on the growth and development of Arabidopsis plants ( Arabidopsis thaliana ecotype Columbia [L.] Heynh) were evaluated. Initially, all leaves were evaluated on plants 18, 22, 26 and 29 days old. The younger leaves were found to exhibit maximal petiole elongation and upward leaf bending in response to BL treatment. Therefore, based on these results leaves 6, 7 and 8 on 22–24-day-old plants were selected for all subsequent studies. Elongation along the length of the petiole in response to BL treatment was uniform with the exception of an approximately 4 mm region next to the leaf where upward curvature was observed. Both BL and 24-epibrassinolide (24-epiBL) were evaluated, with BL being more effective at lower concentrations than 24-epiBL. The exaggerated growth induced by 0.1 μ M BL was not observed in plants treated with 1 000-fold higher concentrations of GA₃, IAA, NAA or 2,4-D (100 μ M ). In addition, no exaggerated growth effects were observed when plants were treated with 200 ppm ethylene or 1 m M ACC. All treatments with BL, NAA, 2,4-D, IAA or ACC promoted ethylene and ACC production in wild type Arabidopsis plants, but only BL triggered exaggerated plant growth. BL also promoted exaggerated growth and elevated levels of ACC and ethylene in the ethylene insensitive mutant etr1-3 , showing that the effect of BR on growth is independent of ethylene. This work provides evidence that BR-induced exaggerated growth of Arabidopsis plants is independent of gibberellins, auxins and ethylene.     

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O₂ caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O₂. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography-mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH⁺₄-fed and 8.3 for NO⁻₃-fed plants) than for N₂-fixing plants (2.5). Significant amounts of ¹⁵N added as ¹⁵NH⁺₄ or ¹⁵NO⁻₃ accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of ¹⁵NH⁺₄ led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.     

Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source

The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m⁻¹, containing nitrogen (0.5 or 4.5 m M ) in the form of NH₄NO₃ or NaNO₃ Salinity-treated (11.2 dS m⁻¹) plants produced less biomass and more organic nitrogen while accumulating more NO⁻₃ than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO⁻₃ accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH₄NO₃ or NaNO₃. Similar effects were observed in roots of plants grown in 4.5 m M NaNO₃. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Influence of bicarbonate and metal ions on the development of root Fe(III) reducing capacity by Fe-deficient cucumber (Cucumis sativus) plants

The effect of bicarbonate and selected metal ions on the development of enhanced root Fe(III) reducing capacity (a response to Fe deficiency of dicotyledons) was studied in young plants of cucumber ( Cucumis sativus L. cv. Ashley) grown in nutrient solution. Pretreatment of 11-day-old Fe-deficient cucumber plants with 20 m M NaHCO₃, for at least 23 h prior to determination of root Fe(III) reducing capacity, markedly inhibited this response. The inhibitory effect of bicarbonate could be partly reversed by a 4- to 8-h treatment with either 10 μ M MnSO₄, 10 μ M FeEDDHA, 2 μ M ZnSO₄, 0.5 μ M NiCl₂, or 0.25 μ M , or CoSO₄ (final concentrations), added to the nutrient solution. By contrast, the addition of other salts of metal ions, like CuSO₄ and Cd(NO₃)₂, at 0.25, 0.5 or 1 μ M , or MgSO₄, at 0.5, 1 or 2 m M (final concentrations), had no beneficial effect. The results suggest that bicarbonate may inhibit the development of root Fe(III) reducing capacity by diminishing the availability of certain metal ions required for this response.