The identification of Fe transport forms in plant xylem sap is crucial to the understanding of long-distance Fe transport processes in plants. Previous studies have proposed that Fe may be transported as an Fe-citrate complex in plant xylem sap, but such a complex has never been detected. In this study we report the first direct and unequivocal identification of a natural Fe complex in plant xylem sap. A tri-Fe(III), tri-citrate complex (Fe(3)Cit(3)) was found in the xylem sap of Fe-deficient tomato (Solanum lycopersicum Mill. cv. 'Tres Cantos') resupplied with Fe, by using an integrated mass spectrometry approach based on exact molecular mass, isotopic signature and Fe determination and retention time. This complex has been modeled as having an oxo-bridged tri-Fe core. A second complex, a di-Fe(III), di-citrate complex was also detected in Fe-citrate standards along with Fe(3)Cit(3), with the allocation of Fe between the two complexes depending on the Fe to citrate ratio. These results provide evidence for Fe-citrate complex xylem transport in plants. The consequences for the role of Fe to citrate ratio in long-distance transport of Fe in xylem are also discussed.

Isotope pattern deconvolution is a mathematical technique for isolating distinct isotope signatures from mixtures of natural abundance and enriched tracers. In iron metabolism studies measurement of all four isotopes of the element by high-resolution multicollector or collision cell ICP-MS allows the determination of the tracer/tracee ratio with simultaneous internal mass bias correction and lower uncertainties. This technique was applied here for the first time to study iron uptake by cucumber plants using 57Fe-enriched iron chelates of the o,o and o,p isomers of ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA) and ethylenediamine tetraacetic acid (EDTA). Samples of root, stem, leaves, and xylem sap, after exposure of the cucumber plants to the mentioned 57Fe chelates, were collected, dried, and digested using nitric acid. The isotopic composition of iron in the samples was measured by ICP-MS using a high-resolution multicollector instrument. Mass bias correction was computed using both a natural abundance iron standard and by internal correction using isotope pattern deconvolution. It was observed that, for plants with low 57Fe enrichment, isotope pattern deconvolution provided lower tracer/tracee ratio uncertainties than the traditional method applying external mass bias correction. The total amount of the element in the plants was determined by isotope dilution analysis, using a collision cell quadrupole ICP-MS instrument, after addition of 57Fe or natural abundance Fe in a known amount which depended on the isotopic composition of the sample.

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were characterised on a multi-collector ICP-MS instrument both in terms of isotopic composition and concentration. The isotopic composition of the spikes was determined using either a natural abundance or certified standard for mass bias correction, except for lead where thallium internal correction was used. It was observed that the use of weighted regression lines for mass bias correction provided similar or, in most cases, lower uncertainties in the isotopic composition of the spikes in comparison with unweighted regression lines or single ratio measurements. For the establishment of the spike concentration by reverse isotope dilution analysis, isotope pattern deconvolution was evaluated and mass bias could be corrected internally in each blend by minimising the variance of the multiple linear regression model. It was also observed that isotope pattern deconvolution with internal mass bias correction provided lower uncertainties in the concentration of the spikes in comparison with the usual procedure based on external mass bias correction.

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