Research with stable isotopes has been mainly developed for the study of the natural variations in the stable isotope abundance of light elements such as H, C and O (Belshaw et al., 2000), as well as for tracing metabolic studies using organic molecules enriched in the heavy isotopes of H, C and N (see review by Patterson and Veillon, 2001). Stable isotopes such as 2H, 13C and 15N have been used in some studies related to plant Fe research. The 13C natural abundance was used to study the changes induced by Fe deficiency in C fixation in sugar beet plants (Rombola et al., 2005). Since Fe-deficient plants have a marked increase in root PEPCase activity (Andaluz et al., 2002), it was hypothesized that an increase in the amount of root C fixation by this enzyme, compared to the amount of C fixed by Rubisco in leaves, would cause higher (less negative) S13C values, as a result of the different degree of C discrimination of these two enzymes (AS13C of-29%0 for Rubisco and -6%o for PEPCase; Lajtha and Marshall, 1994). However, S13C was instead higher (less negative) in Fe-sufficient than in Fe-deficient plants, and the reason for this finding is still unexplained. In the same study, nutrient solution HCO3- was labeled with 13C as a tracer to quantify the contribution of the C deriving from bicarbonate to total plant C (Rombola et al., 2005). Hydrogen-2, 13C and 15N have also been used to characterize the biosynthetic pathway of mugineic acids, a group of well-known phyto-siderophores produced by graminaceous species in response to Fe stress (Ma and Nomoto, 1994; Ma et al., 1995). Tracing experiments were conducted by feeding roots with several metabolites labelled in different positions with 2H, 13C and 15N. The incorporation of these labelled atoms into mugineic acids was studied by using NMR (1H, 2H and 13C) and fast atom bombardment (FAB; 2H and 15N) mass spectrometry. From these experiments it was concluded that the methionine recycling pathway was involved not only in ripening processes, but was also required for continued synthesis of mugineic acids in roots.
Carbon radioisotopes such as 14C and 11C have also been used in plant Fe research. Carbon-14 was used to study the plant uptake and breakdown of synthetic Fe(III) chelates by Hill-Cottingham and Lloyd-Jones (1961) and Jeffreys et al. (1961), using 14C labelled EDTA and EDDHA to synthesize 59Fe-14C-labeled Fe(III)-EDTA and -EDDHA. These dual-labelled Fe-chelates were used to investigate the uptake of the whole organic molecule by roots (Hill-Cottingham and Lloyd-Jones, 1961). Recently, a protocol to incorporate 14C into nicotianamine, a non-proteinogenic amino acid considered to be a key component in Fe homeostasis in plants, has been developed by Schmiedeberg et al. (2003), using the precursor S-adenosylmethionine labelled with 14C and a recombinant nicotianamine synthase. Also, studies with 14C were made to identify maize root exudates involved in Fe trafficking in the rhizosphere and to estimate their amount (Kuzyakov et al., 2003). Maize plants were labelled in a 14CO2 atmosphere to separate root-derived and soil-derived organic substances in the rhizosphere using a nonsterile soil, and secreted compounds present in lixiviation fluids were analyzed for total 14C radioactivity and also for composition using pyrolysis field ionization-mass spectrometry. The half-life of mugineic acid secreted by roots of barley grown in 14CO2-enriched atmosphere was found to be approximately 24 hours (Mori et al., 1987). Working with barley, positron (y-ray) 11C emission imaging has been used to investigate whether methio-nine transported from the shoot was used for the direct synthesis of mugineic acids in the roots of Fe-deficient plants (Nakanishi et al., 1999).
Was this article helpful?