Ethylene involvement in the over-expression of Fe(III)-chelate reductase by roots of E107 pea [Pisum sativum L. (brz, brz)] and chloronerva tomato (Lycopersicon esculentum L.) mutant genotypes

Recently, ethylene was reported to be involved in the regulation of Fe(III)-chelate reducing capacity by cucumber (Cucuinis sativus L.) roots. Here, we studied the effect of two ethylene inhibitors, aminooxyacetic acid (AOA) and cobalt, on the Fe(III) reducing capacity in roots of mutant genotypes [E107 pea [Pisum sativum L. (brz, brz)] and chloronerva tomato (Lycopersicon esculentum L.) that exhibit high rates of Fe(III)-chelate reduction and excessive iron accumulation. The ethylene inhibitors, AOA and cobalt, markedly inhibited Fe(III)-chelate reducing capacity in roots of both genotypes. Over-expression of root Fe(III) reductase activity by both mutants appears to be related to ethylene. Possibly, both mutants are genetically defective in their ability to regulate root ethylene production. The large inhibitory effect of both ethylene inhibitors on Fe(III)-chelate reducing capacity in roots of the mutant tomato genotype, chloronerva, disputes the contention that the nicotianamine-Fe(II) complex is the repressior of the gene responsible for Fe(III)-chelate reductase activity, as previously suggested by others. However, since nicotianamine shares the same biosynthetic precursor as ethylene, i.e. S-adenosyl methionine, nicotianamine may affect Fe(III)-chelate reductase activity in dicot and non-grass monocot roots by influencing ethylene biosynthesis.     

Shoot-to-Root Signal Transmission Regulates Root Fe(III) Reductase Activity in the dgl Mutant of Pea

To understand the root, shoot, and Fe-nutritional factors that regulate root Fe-acquisition processes in dicotyledonous plants, Fe(III) reduction and net proton efflux were quantified in root systems of an Fe-hyperaccumulating mutant (dgl) and a parental (cv Dippes Gelbe Viktoria [DGV]) genotype of pea (Pisum sativum). Plants were grown with (+Fe treated) or without (-Fe treated) added Fe(III)-N,N'-ethylenebis[2-(2-hydroxyphenyl)-glycine] (2 [mu]M); root Fe(III) reduction was measured in solutions containing growth nutrients, 0.1 mM Fe(III)-ethylenediaminetetraacetic acid, and 0.1 mM Na2-bathophenanthrolinedisulfonic acid. Daily measurements of Fe(III) reduction (d 10-20) revealed initially low rates in +Fe-treated and -Fe-treated dgl, followed by a nearly 5-fold stimulation in rates by d 15 for both growth types. In DGV, root Fe(III) reductase activity increased only minimally by d 20 in +Fe-treated plants and about 3-fold in -Fe-treated plants, beginning on d 15. Net proton efflux was enhanced in roots of -Fe-treated DGV and both dgl growth types, relative to +Fe-treated DGV. In dgl, the enhanced proton efflux occurred prior to the increase in root Fe(III) reductase activity. Reductase studies using plants with reciprocal shoot:root grafts demonstrated that shoot expression of the dgl gene leads to the generation of a transmissible signal that enhances Fe(III) reductase activity in roots. The dgl gene product may alter or interfere with a normal component of a signal transduction mechanism regulating Fe homeostasis in plants.     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.     

Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity

All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.     

Direct Measurement of 59Fe-Labeled Fe2+ Influx in Roots of Pea Using a Chelator Buffer System to Control Free Fe2+ in Solution

Fe2+ transport in plants has been difficult to quantify because of the inability to control Fe2+ activity in aerated solutions and non-specific binding of Fe to cell walls. In this study, a Fe(II)-3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4[prime]4"-disulfonic acid buffer system was used to control free Fe2+ in uptake solutions. Additionally, desorption methodologies were developed to adequately remove nonspecifically bound Fe from the root apoplasm. This enabled us to quantify unidirectional Fe2+ influx via radiotracer (59Fe) uptake in roots of pea (Pisum sativum cv Sparkle) and its single gene mutant brz, an Fe hyperaccumulator. Fe influx into roots was dramatically inhibited by low temperature, indicating that the measured Fe accumulation in these roots was due to true influx across the plasma membrane rather than nonspecific binding to the root apoplasm. Both Fe2+ influx and Fe translocation to the shoots were stimulated by Fe deficiency in Sparkle. Additionally, brz, a mutant that constitutively exhibits high ferric reductase activity, exhibited higher Fe2+ influx rates than +Fe-grown Sparkle. These results suggest that either Fe deficiency triggers the induction of the Fe2+ transporter or that the enhanced ferric reductase activity somehow stimulates the activity of the existing Fe2+ transport protein.     

Sulphur deprivation limits Fe-deficiency responses in tomato plants

The aim of this work was to clarify the role of S supply in the development of the response to Fe depletion in Strategy I plants. In S-sufficient plants, Fe-deficiency caused an increase in the Fe(III)-chelate reductase activity, ⁵⁹Fe uptake rate and ethylene production at root level. This response was associated with increased expression of LeFRO1 [Fe(III)-chelate reductase] and LeIRT1 (Fe²⁺ transporter) genes. Instead, when S-deficient plants were transferred to a Fe-free solution, no induction of Fe(III)-chelate reductase activity and ethylene production was observed. The same held true for LeFRO1 gene expression, while the increase in ⁵⁹Fe²⁺ uptake rate and LeIRT1 gene over-expression were limited. Sulphur deficiency caused a decrease in total sulphur and thiol content; a concomitant increase in ³⁵SO₄ ²⁻ uptake rate was observed, this behaviour being particularly evident in Fe-deficient plants. Sulphur deficiency also virtually abolished expression of the nicotianamine synthase gene (LeNAS), independently of the Fe growth conditions. Sulphur deficiency alone also caused a decrease in Fe content in tomato leaves and an increase in root ethylene production; however, these events were not associated with either increased Fe(III)-chelate reductase activity, higher rates of ⁵⁹Fe uptake or over-expression of either LeFRO1 or LeIRT1 genes. Results show that S deficiency could limit the capacity of tomato plants to cope with Fe-shortage by preventing the induction of the Fe(III)-chelate reductase and limiting the activity and expression of the Fe²⁺ transporter. Furthermore, the results support the idea that ethylene alone cannot trigger specific Fe-deficiency physiological responses in a Strategy I plant, such as tomato.     

Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper

The Arabidopsis FRO2 gene encodes the iron deficiency-inducible ferric chelate reductase responsible for reduction of iron at the root surface; subsequent transport of iron across the plasma membrane is carried out by a ferrous iron transporter (IRT1). Genome annotation has identified seven additional FRO family members in the Arabidopsis genome. We used real-time RT-PCR to examine the expression of each FRO gene in different tissues and in response to iron and copper limitation. FRO2 and FRO5 are primarily expressed in roots while FRO8 is primarily expressed in shoots. FRO6 and FRO7 show high expression in all the green parts of the plant. FRO3 is expressed at high levels in roots and shoots, and expression of FRO3 is elevated in roots and shoots of iron-deficient plants. Interestingly, when plants are Cu-limited, the expression of FRO6 in shoot tissues is reduced. Expression of FRO3 is induced in roots and shoots by Cu-limitation. While it is known that FRO2 is expressed at high levels in the outer layers of iron-deficient roots, histochemical staining of FRO3-GUS plants revealed that FRO3 is predominantly expressed in the vascular cylinder of roots. Together our results suggest that FRO family members function in metal ion homeostasis in a variety of locations in the plant.     

The effect of sodium bicarbonate on plant performance and iron acquisition system of FA-5 (Forner-Alcaide 5) citrus seedlings

This work studies the effect of bicarbonate on plant performance and the iron acquisition system of Forner-Alcaide 5 (FA-5) seedlings, a citrus genotype known for its tolerance to calcareous soils. Plants were irrigated for 6 weeks with or without 10 mM NaHCO₃. Treatment significantly decreased shoot growth, photosynthetic levels and iron concentration in shoots and roots. o,o-⁵⁷FeEDDHA experiments indicated that ⁵⁷Fe uptake by roots was inhibited in treated plants. Moreover, those seedlings accumulated more ⁵⁷Fe in roots, and enhanced mRNA accumulation of ferric reductase genes FRO1 and FRO2 and FC-R activity in roots. H⁺-ATPase activity and HA1 gene expression were also increased, while HA2 was not affected. In addition, expression of the iron transporter gene IRT1 was increased, while IRT2 was not significantly affected. Finally, according to PEPC enzymatic activity, PEPC1 gene expression was higher in treated roots. In conclusion, it appears that bicarbonate prevents medium acidification by roots, thus reducing Fe²⁺ uptake. Accordingly, Fe deficiency enhanced the expression of some genes related with the Fe acquisition system (IRT1, FRO1, FRO2, HA1 and PEPC1) and the activity of the corresponding enzymes, which appear to constitute an adaptive mechanism of FA-5 in these soils.     

A copper-deficiency-induced root reductase is different from the iron-deficiency-induced one in red clover (Trifolium pratense L.)

There is increasing evidence that Cu deficiency can induce root reductase activity, but the ecological and physiological significance of this is unknown. This study compared the characteristics of root reductase activity induced by Cu deficiency with those induced by Fe deficiency in red clover (Trifolium pratenseL. cv. Kenland), a Fe-efficient plant. Effects of other nutritional stresses were also investigated for comparison. Compared with the effect of Fe deficiency, Cu deficiency induced only a moderate level of root reductase activity, while other nutrient stresses had no effect, or even inhibited the root reductases activity, especially in the case of Zn deficiency. Compared with Fe deficiency-induced Fe(III)-chelate reductase, Cu deficiency-induced reductase displayed a different pattern of induction. The activity of the Cu deficiency-induced reductase in intact plants increased with time; in decapitated plants it showed a distinct peak at a later stage of the treatment. The Fe concentration in the roots was significantly increased under Cu deficiency. Furthermore, the reductase activity was presented in the entire root system, contrary to what was observed for the Fe-deficiency-induced reductase activity, which was confined to the root apex. Cu deficiency did not increase proton extrusion from the roots, even when growth was significantly affected. The present results suggest that in red clover Cu deficiency induces a root reductase that is different from the reductase induced by Fe deficiency.     

Light-regulated, tissue-specific, and cell differentiation-specific expression of the Arabidopsis Fe(III)-chelate reductase gene AtFRO6

Iron is an essential element for almost all living organisms, actively involved in a variety of cellular activities. To acquire iron from soil, strategy I plants such as Arabidopsis (Arabidopsis thaliana) must first reduce ferric to ferrous iron by Fe(III)-chelate reductases (FROs). FRO genes display distinctive expression patterns in several plant species. However, regulation of FRO genes is not well understood. Here, we report a systematic characterization of the AtFRO6 expression during plant growth and development. AtFRO6, encoding a putative FRO, is specifically expressed in green-aerial tissues in a light-dependent manner. Analysis of mutant promoter-beta-glucuronidase reporter genes in transgenic Arabidopsis plants revealed the presence of multiple light-responsive elements in the AtFRO6 promoter. These light-responsive elements may act synergistically to confer light responsiveness to the AtFRO6 promoter. Moreover, no AtFRO6 expression was detected in dedifferentiated green calli of the korrigan1-2 (kor1-2) mutant or undifferentiated calli derived from wild-type explants. Conversely, AtFRO6 is expressed in redifferentiated kor1-2 shoot-like structures and differentiating calli of wild-type explants. In addition, AtFRO7, but not AtFRO5 and AtFRO8, also shows a reduced expression level in kor1-2 green calli. These results suggest that whereas photosynthesis is necessary but not sufficient, both light and cell differentiation are necessary for AtFRO6 expression. We propose that AtFRO6 expression is light regulated in a tissue- or cell differentiation-specific manner to facilitate the acquisition of iron in response to distinctive developmental cues.     

Possible role of root-ethylene in Fe(III)-phytometallophore uptake in Strategy II species

All phytometallophores are derived from methionine through S-adenosylmethionine (SAM) via nicotianamine. Ethylene is synthesized from methionine via SAM and 1-aminocyclopropane-1-carboxylic acid (ACC). This close similarity in biochemical pathways suggests that root-ethylene may play a role in regulating Fe(III)-phytometallophore homeostasis in cereal (Strategy II species) roots as well as in the regulation of Fe(III)-chelate reductase activity in Strategy I species. Barley (Hordeum vulgare L.) seedlings were grown in chelate-buffered nutrient solutions with increasing levels of Fe (i.e., 5, 25 or 100 µM Fe) as Fe(III)-HEDTA. Seedlings at each level of Fe were treated with either an inhibitor or a promoter of ethylene action. Treatment with the promoter, ACC (1 µM), had no significant effect of phytometallophore root efflux or Fe uptake by 19-d-old barley seedlings at all Fe levels. However, treatment with the inhibitor, aminooxyacetic acid (AOA, 10 µM) repressed the ability of cereal roots to absorb sufficient Fe to meet metabolic needs, but surprisingly enhanced phytometallophore root efflux rates at all Fe(III)-HEDTA levels. These results support a possible role of root-ethylene in Fe(III) uptake in cereals, but the mechanism remains unclear.     

Technical advance: reduction of Fe(III)-chelates by mesophyll leaf disks of sugar beet. Multi-component origin and effects of Fe deficiency

The characteristics of the Fe(III)-chelate reductase activity have been investigated in mesophyll disks of Fe-sufficient and Fe-deficient sugar beet leaves. The Fe(III)-chelate reductase activity of mesophyll disks was light dependent and increased markedly when the epidermis was removed. Iron(III)-citrate was photo-reduced directly by light in the absence of plant tissue. Total reductase activity was the sum of enzymatic mesophyll reduction, enzymatic reduction carried out by organelles exposed at the disk edge and reduction caused by the release of substances both by exposed mesophyll cells and at the disk edge. Compounds excreted were shown by HPLC to include organic anions, mainly oxalate, citrate and malate. When expressed on a leaf surface basis, Fe deficiency decreased the total mesophyll Fe(III)-chelate reductase activity. However, Fe-sufficient disks reduced less Fe than the Fe-deficient ones when expressed on a chlorophyll basis. The optimal pH values for Fe(III) reduction were always in the range 6.0-6.7. In control leaves Fe(III)-citrate and Fe(III)-malate were the substrates that led to the highest Fe reduction rates. In Fe-deficient leaves Fe(III)-malate led to the highest Fe reduction rates, followed by Fe(III)-EDTA and then Fe(III)-citrate. K:(m) values for the total reductase activity, enzymatic mesophyll reduction and enzymatic reduction carried out by organelles at the disk edge were obtained.     

Reduction of root iron in Plantago lanceolata during recovery from Fe deficiency

Changes in root-associated Fe(III) reductase activity and Fe concentration during recovery from temporary iron starvation were investigated in hydroponically grown Plantago lanceolata L. Within two days, interruption of the Fe supply resulted in enhanced rates of reduction by intact plant roots. Transfer of iron deficient plants to a solution containing various amounts of chelated Fe caused a transient increase in reduction activity before the rates declined to a level determined by the amount of Fe added. Repression of root-associated redox activity was independent of the Fe concentration in the preculture. When iron deficient plants were submitted to a supply of Fe localized to a part of the root system (split-root plants), the decrease in reduction rates was much more pronounced in the Fe-deprived portion of the roots than in the Fe-supplied one. No correlation was observed between root Fe concentration and Fe(III) reductase activity. Continued growth of split-root plants in the +Fe/-Fe regime increased the reduction rates of the +Fe-grown portion of the root system over the rates in iron sufficient plants with non-divided roots. The results are discussed in relation to putative factors mediating intra- and interorgan regulation of iron nutrition.