Tracer studies and several plant mutants have provided an overview of the route of Fe from uptake at the root surface to the vascular system and back from leaves to the vasculature to sink organs (reviewed in Hell and Stephan, 2003). Iron allocation seems to be a two-step process with first transport in the xylem followed by remobilization via the phloem. For example, when Fe is supplied directly to the xylem it is first transported to the leaves but not to the apex as the most obvious sink to maintain growth (Zhang et al., 1995). Furthermore, Fe applied to leaves follows the symplastic route and the phloem (Edding and Brown, 1967). This detour is probably caused by incomplete xylem structures in growing organs and their small transpiration rates resulting in little water transport. This has a major impact on plant growth under Fe deficiency, where young leaves suffer more than mature leaves, but also on crop quality, since fruits and storage organs are almost exclusively dependent on Fe import from the phloem. Thus, improving the Fe content of staple food seeds, which is urgently needed in large areas of the world, is a multi-step process difficult to achieve by means of breeding and biotechnology (Grusak and Della Penna, 1999). Unfortunately, our knowledge on the biochemical and molecular processes responsible for the exchange of chelated Fe between these numerous different tissue types is still very fragmentary.
In the xylem, the majority of Fe is complexed as ferric citrate and presumably transported as such. Precise mechanisms for loading of xylem with Fe or ferric citrate and its unloading in leaves have not yet been described.
During germination, the growing shoot apex receives Fe from cotyledons through the phloem, providing a more accessible experimental system for analysis of phloem transport than mature plants. Feeding of 55Fe and 65Zn demonstrated that the vast majority of Fe was associated with the protein fraction in the phloem, while Zn was bound to low-molecular weight compounds. An Fe transport protein (RcITP) was isolated biochemically from Ricinus communis cotyledon phloem sap, due to its capability to bind Fe (Krüger et al., 2002). In vitro, ITP binds preferentially Fe(III) over Fe(II) but it also forms complexes with Zn, Cu and Mn. One protein molecule is able to bind several metal ions. This was corroborated by the amino acid sequence, which consists mostly of polar residues. Homology searches showed that RcITP belongs to the family of late embryogenesis abundant proteins (LEA) and in particular to the group II called dehydrins (Close, 1996). Dehydrins are highly hydrophilic, stay soluble during heating and are generally expressed during later stages of seed development as well as in other organs in response to drought, cold or high salinity (Nylander et al., 2001). Indeed, a protein with strong sequence similarity to RcITP was found in the Arabidopsis genome sequence (AtITP; MIPS code At1g54410). Recombinant AtITP is able to bind Fe(III) and also Ni in comparable ways as RcITP, suggesting an ortholog relationship of the two proteins (C. Krüger, R. Hell, unpublished). Expression patterns of RcITP and AtITP genes reveal differential expression levels, but nearly ubiquitous presence in tissues with vasculature (Krüger et al., 2002; N. Piening, M. Gahrtz, R. Hell, unpublished).
These findings strongly suggest that ITP as a component of long-distance transport of Fe (and possibly other micronutrients). The conditions that lead to loading of Fe onto ITP in the phloem and unloading at target organs remain unclear. Recently, other members of the dehydrin family have been shown to bind Ca in vitro. Interestingly, the Ca binding properties of these proteins are modulated by phosphorylation (Alsheikh et al., 2002; Heyen et al., 2002). It cannot be excluded that this or another post-translational modification mechanism controls loading and unloading of ITP, but chemical conditions such as pH and ion concentration could also affect the number of ions bound per ITP protein.
The metal chelator nicotianamine is transported at long distance in the plant and is able to bind Fe and other metals (Scholz et al., 1992). Nicotianamine plays a key role in intercellular and intracellular transport of Fe that is still not fully elucidated, and may contribute to xylem and phloem loading and unloading of Fe, as well as long-distance Fe transport (for review see Hell and Stephan, 2003). Nicotianamine is a plant-specific, non-proteinogenic amino acid that derives from the condensation of 3 molecules of S-adenosyl-L-methionine through the action of nicotianamine synthase (Herbik et al., 1999; Higuchi et al., 1999; Ling et al., 1999). Iron deficiency in the nicotianamine-free tomato mutant chloronerva results in chlorotic interveinal leaf areas, hence the name of the mutant. Since chloronerva plants still have Fe in their leaves, nicotianamine does not seem to be essential for all these processes (Scholz et al., 1992; Takahashi et al., 2003). However, the biochemical observations of preferred binding of Fe(III) by ITP and a more stable complex of Fe(II) with nicotianamine raise the possibility of functional, or even physical, interaction of the protein and the chelator in the phloem, in order to mediate transport or loading processes (Krüger et al., 2002; von Wiren et al., 1999). Another interesting observation related to nicotianamine was that nicotianamine synthase genes from rice were expressed in proximity of vascular tissues, suggesting that indeed these cells may produce nicotianamine for regulating long-distance transport (Inoue et al, 2003). Moreover, AtYSL2 and OsYSL2 (YSL = YELLOWSTRIPE-like) genes encoding transporter homologs of ZmYSl are also expressed in the vascular strands, in proximity to the phloem and xylem (DiDonato et al., 2004; Koike et al., 2004). AtYSL2 was found induced by Fe supply in roots and leaves and repressed upon Fe deficiency (DiDonato et al., 2004). It was speculated that AtYSL2 may prevent exit of xylem Fe into roots and mature leaves upon Fe deficiency to allow better Fe nutrition of young shoots (DiDonato et al., 2004). Recent studies by Schaaf et al. (2005) suggested however that AtYSL2 might not be involved in iron-nicotianamine transport. In contrast, OsYSL2 was induced by Fe deficiency in leaves and then expressed not only in the vascular strands but in nearly all tissues (Koike et al. , 2004). Nicotianamine is also involved in intercellular and intracellular transport and its possible function is discussed further in the following paragraph.
It is an interesting question how Fe loading into and unloading from the vascular system is regulated. The basic helix-loop-helix regulator protein FER is a transcription factor that controls Fe uptake in the root. The FER gene is expressed in a developmental pattern along the root. FER is not only expressed in the epidermis of the elongation zone, but also in parenchyma cells of the vascular root cylinder in the root hair zone (Ling et al., 2002). FER may regulate in these latter parenchymatic cells uptake and transport of Fe. It is also possible; however, that the FER protein delivers or responds to an Fe signal from the shoot. Interestingly, these same parenchymatic cells expressed the Fe transporter genes LeIRT2 and LeNRAMPl (Bereczky et al., 2003). LeIRT2, although similar in sequence to LeIRTI, is not Fe-regulated and expressed irrespective of the FER genotype (Bereczky et al., 2003; Eckhardt et al., 2001). LeIRT2 may function in a different aspect of metal transport. LeNRAMPl expression is partially dependent on FER (Bereczky et al, 2003). A homolog of LeNRAMPl, AtNRAMP3, is also expressed in the vascular cylinder in Arabidopsis and may serve the mobilisation of intracellular Fe pools (Thomine et al., 2003). AtFRD3, encoding a putative oligopeptide transporter of unknown function, is expressed in the root pericycle and vascular cylinder (Green and Rogers, 2004; Rogers and Guerinot, 2002). Chlorotic frd3 mutant plants over-accumulate Fe in the vascular cylinder of the root (Green and Rogers, 2004). FRD3 therefore seems to act in root xylem loading or in loading of factors for Fe transport towards the leaves.
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