Iron uptake system mediates nitrate-facilitated cadmium accumulation in tomato (Solanum lycopersicum) plants

Nitrogen (N) management is a promising agronomic strategy to minimize cadmium (Cd) contamination in crops. However, it is unclear how N affects Cd uptake by plants. Wild-type and iron uptake-inefficient tomato (Solanum lycopersicum) mutant (T3238fer) plants were grown in pH-buffered hydroponic culture to investigate the direct effect of N-form on Cd uptake. Wild-type plants fed NO?? accumulated more Cd than plants fed NH??. Iron uptake and LeIRT1 expression in roots were also greater in plants fed NO??. However, in mutant T3238fer which loses FER function, LeIRT1 expression in roots was almost completely terminated, and the difference between NO?? and NH?? treatments vanished. As a result, the N-form had no effect on Cd uptake in this mutant. Furthermore, suppression of LeIRT1 expression by NO synthesis inhibition with either tungstate or L-NAME, also substantially inhibited Cd uptake in roots, and the difference between N-form treatments was diminished. Considering all of these findings, it was concluded that the up-regulation of the Fe uptake system was responsible for NO??-facilitated Cd accumulation in plants.     

pH Changes Associated with Iron-Stress Response

When Fe-inefficient T3238fer and Fe-efficient T3238FER tomatoes were supplied iron, and nitrogen as nitrate, they increased the pH of the nutrient culture. When they were supplied nitrogen as ammonium, they decreased the pH. When Fe supply was limited, Fe-stress response developed in T3238FER that opposed the usual nitrate response and decreased, rather than increased, the pH. A “reductant” which reduced Fe³⁺ to Fe²⁺ was released from the roots of these plants and lowered the pH; and there was a tremendous increase in the uptake of Fe. T3238fer did not produce “reductant” in response to Fe-stress; the pH increased, and the plants developed Fe-deficiency when nitrogen was supplied as nitrate. Nitrogen nutrition and iron-stress response are important factors associated with iron chlorosis in plants. Release of hydrogen ions from roots of Fe-stressed plants is caused by more than response to imbalanced uptake of cations and anions.     

Iron-Stress Response in Tomato (Lycopersicon esculentum) 1. Sites of Fe Reduction, Absorption and Transport

T3238fer (Fe-inefficient) and T3238FER (Fe-efficient) tomato plants differ in their ability to utilize Fe and therefore can be used as test genotypes to locate sites of Fe uptake or to characterize changes that occur in roots in response to Fe stress (Fe deficiency). T3238fer does not respond to Fe stress. Release of hydrogen ions and reduction of Fe³⁺ to Fe²⁺ are two primary responses of T3238FER roots to Fe stress. Fe reduction sites were predominately in the young lateral roots, and between the regions of root elongation and maturation of the primary root. The use of BDPS (bathophenanthrolinedisulfonate) to trap Fe²⁺ did not affect the release of H⁺ ions or reduction by T3238FER roots. BPDS did not decrease Fe uptake until it exceeded the Fe concentration in the nutrient solution. A sevenfold increase in BPDS caused a threefold decrease in Fe taken up by the plant. Fe³⁺ is reduced to Fe²⁺ at root sites accessible to BPDS. Adding Zn decreased the response to Fe stress. Iron stress initiates the development of lateral roots, and we propose that most Fe enters the plant through these roots. The iron moves through protoxylem into the metaxylem of the primary root and then to the top of the plant as Fe citrate. Root environmental factors that are competitive or inhibit Fe-stress response, or genotypes that fail to respond to Fe stress, contribute to the development of Fe deficiency in plants.     

Effect of Pseudomonas fluorescens on the root exudates of two tomato mutants differently sensitive to Fe chlorosis

Plants with different Fe-mobilization properties are known to differ in the amount and kind of Fe-reducing and Fe-chelating compounds exuded by their roots. Although rhizosphere bacteria are known to affect the exudation of organic compounds by the plant roots, their effect on the root exudates of plants differing in Fe-mobilization properties is not known. We studied the effect of Pseudomonas fluorescens, on the exudation of sugars and organic and amino acids by roots of an iron chlorosis-resistant (T3238FER) and a chlorosis-susceptible (T3238fer) tomato mutant. Under sterile conditions two tomato mutants grew equally well and did not differ in the total amount of sugars and organic acid exuded by their roots. More amino acids, however, were exuded by the roots of T3238FER than T323fer. Mutants differed in the amount of oxalic acid and the amino acids Ala, Asp, Gaba, Gln, Gly, His, Hyl, Ile, Leu, Lys, Phe, Pro, and Val exuded by their roots into sterile rooting media. Addition of P. fluorescens to the rooting medium did not affect the growth of T3238FER but stimulated the root growth of chlorosis-susceptible T3238fer, reduced the amounts of glucose, arabinose and fructose but increased the amount of sucrose, reduced the amounts of fumaric, malic and oxalic acid but increased the amounts of citric and succinic acid in the rooting media of both mutants. P. fluorescens resulted in the following changes in the amino acids in the rooting media: reduced the amounts of Gly, Leu, and Lys in T3238FER, and of Asp, Gln, Hyp, and Ile in T3238fer, and increased the amounts of Cys, Glu, His, Hyp, Ile, Phe and Tyr in T3238FER and of Ala, Glu, His, Phe, and Ser in T323fer—in cases more than 40-fold. These differential effects of P. fluorescens in altering the pattern of organic and amino acids compounds with some Fe-chelating properties detected in the rooting medium of these two mutants may indicate that the differences in Fe-chlorosis susceptibility of these tomato mutants may be the result of, or modified by, the interactions between plant roots and rhizosphere microorganisms. We postulate that the Fe-chlorosis susceptibility in plants may be the product of the interactions between soil microorganisms and plant roots, and may not be solely related to the plant per se.     

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.     

Sequence Diversity and Enzyme Activity of Ferric-Chelate Reductase LeFRO1 in Tomato

Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron ho- meostasis in strategy I plants. LeFROI is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 （LeFRO1MM, LeFRO1Ailsa and LeFRO1Monita） with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity （LeFRO1AiISa 〉 LeFRO1MM 〉 LeFRO1M~nita）. The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue lie at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.     

Nitrate Reductase Activity in Calcifugous and Calcicolous Tomatoes as Affected by Iron Stress

Calcicolous plants are generally more Fe-efficient than calcifugous plants, because they respond to Fe stress by releasing H-ions and “reductants” from their roots that causes Fe to become available. The objective of our study was to determine if differential response to Fe stress in calcicolous and calcifugous varieties affects nitrate reductase activity. T3238FER (Fe-efficient) and T3238fer (Fe-inefficient) tomato (Lycopersicon esculentum Mill.) cultivars were grown in nutrient solutions supplied with N as NH₄⁺-N plus NO₃^?-N, and as NO₃^?-N only. The chemical reactions induced by Fe stress concomitantly increased nitrate reductase activity in roots and tops of calcicolous, but not in calcifugous tomato. This nitrate reductase activity decreased, however, when Fe was made available to the plants. When Fe stress was eliminated by adding Fe, nitrate reductase activity was comparable in the two cultivars.     

Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato

Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.     

Proteins under the Control of the Gene for Fe Efficiency in Tomato

Fe-deficient dicotyledons develop Fe-efficiency reactions, such as proton extrusion and ferric chelate reduction activity, which are located in the plasma membranes of the root epidermal cells. The fer mutant of tomato (Lycopersicon esculentum Mill.) cannot develop these reactions. Membranes were isolated from roots of wild-type (FER) and mutant (fer) tomato plants grown on nutrient solution with high and low Fe concentrations. Two proteins were identified which are synthesized under the control of the FER gene.