We present evidence suggesting that this Cd over-accumulation may be the result of an enhanced transport of Cd through the plant, making opt3-2 a suitable background for studying long-distance transport of non-essential heavy metals. We further show that OPT3 is targeted to the plasma membrane and is preferentially expressed in the phloem. The Fe/Zn/Mn uptake transporter IRT1 and other ironstarvation-induced genes are constitutively up-regulated in opt3-2. Interestingly, shoot-specific expression of OPT3 restores metal homeostasis and IRT1 up-regulation in roots showing that OPT3 is the first identified molecular component of the network transferring information on the iron status from leaves to roots. Moreover, Fe mobilization between leaves is impaired in opt3-2, suggesting that OPT3 mediates the movement of Fe out of the leaves, and this transport is required for proper communication between leaves and roots and maintenance of the trace-metal homeostasis in Arabidopsis. Understanding phloem-mediated signaling, transport, and seed-loading mechanisms of both essential and non-essential heavy metals will help to develop strategies for excluding toxic metals from seeds and enhance the nutritional value of grains and plant-based products.Members of the Arabidopsis oligopeptide transporter family have been shown to mediate the transport of a broad spectrum of peptides .
Glutathione and phytochelatins are peptides that mediate tolerance and long-distance transport of heavy metals ; therefore, we screened mutants in the Arabidopsis OPT family for differential accumulation of Cd in seeds. A mutant of the Arabidopsis OPT3 gene, opt3-2,plants in pots ideas showed the strongest over-accumulation of Cd in seeds . To test whether this Cd over-accumulation had an effect on seedling growth, assays were performed on plates in the presence and absence of Cd. Figure 1B shows that opt3-2 is hypersensitive to Cd when grown on medium containing 50 μM CdCl2. To determine whether the increased Cd concentration in opt3-2 seeds was due to a systemic over accumulation of Cd throughout the plant, opt3-2 seedlings were grown hydroponically for 6 weeks, exposed to 20 μM CdCl2 for 72h and the metal concentration of roots and leaves was measured by ICP–OES . The roots of opt3-2 over-accumulated Cd compared to wild-type; however, unexpectedly, Cd concentrations in leaves were almost five-fold less than those of wild-type plants . Conversely, seeds of opt3-2 plants show a large increase in Cd levels compared to wild-type seeds .Previous GUS staining experiments have shown that OPT3 is expressed throughout the vasculature; however, localization at a higher resolution has not been evaluated . To identify where in the vasculature OPT3 is preferentially expressed, β-glucuronidase was expressed under the control of the native OPT3 promoter. Under standard growth conditions, GUS staining was negligible; however, under Fe-limiting conditions , staining was clearly observed in the phloem, but not in the pith or endodermis . Consistent with our findings, cell-type-specific micro-array data sets show the highest intensity values of OPT3 in the phloem, comparable to the phloem sucrose transporter SUC2 . Thus, two independent approaches show preferential expression of OPT3 in the phloem.
To gain insight into the subcellular localization of OPT3, an N-terminal YFP–OPT3 translational fusion was infiltrated into Nicotiana benthamiana leaves. Fluorescence was detected along the cell periphery, indicative of plasma membrane localization . A weaker perinuclear fluorescence and transvacuolar strands were also observed in some cells , indicating that a fraction of the YFP–OPT3 localizes to the endoplasmic reticulum . The ER fluorescence pattern, however, was not present in all cells. Furthermore, Hechtian strands were clearly present connecting the cell wall to the plasma membrane of plasmolyzed leaf cells . These results suggest that OPT3 is a plasma membrane transporter preferentially expressed in the phloem.The Arabidopsis mutant opt3-2 shows a constitutive Fe-deficiency response in roots including the up-regulation of the Fe/Zn/Mn transporter IRT1 . Despite this Fe-deficiency response, Fe sensing in shoots remains intact . The molecular mechanisms mediating shoot-to-root signaling of iron status in plants remain largely unknown. The impaired iron sensing in roots but not shoots of opt3-2, in conjunction with phloem localization, suggests a possible role of OPT3 in shoot-to-root transport of a signal reporting metal status. To test this hypothesis, the OPT3 coding sequence was expressed in opt3-2 under the control of the shoot-specific chlorophyll a/b binding protein promoter . Shoot specificity of the CAB2 promoter was determined by GUS staining . RT–PCR analyses confirmed that OPT3 is preferentially expressed in the shoots of three independent transgenic lines . The residual OPT3 transcript in opt3-2 roots expressing CAB2pro:OPT3 plants is consistent with the knockdown nature of the opt3-2 allele. Thus, the low level of OPT3 transcript in roots is not sufficient to properly regulate metal homeostasis in roots .
Two of the major phenotypes described in opt3-2 are the constitutive iron-deficiency response in roots, as illustrated by high IRT1 expression , and the over-accumulation of Cd in seeds . Thus, we tested whether shoot-specific expression of OPT3 was able to complement both phenotypes. As shown by RT– PCR, IRT1 transcript levels were greatly reduced in the roots of CAB2pro:OPT3-expressing plants compared to the opt3-2 mutant . These results show that shoot-specific expression of OPT3 is sufficient for proper regulation of metal homeostasis, including communication between leaves and roots. Furthermore, the Cd accumulation in CAB2pro:OPT3 seeds was reduced to wild-type levels . Seedling hypersensitivity to Cd was also rescued in the three independent CAB2pro:OPT3– expressing lines . Collectively, these results demonstrate that shoot-specific OPT3 expression is sufficient to complement opt3-2 root phenotypes, suggesting that OPT3 may mediate the long-distance transport of a signaling molecule from leaves to relay information about metal status, thus contributing to whole-plant metal homeostasis.To test whether OPT3 functions in the mobilization of Fe or other molecules, we first assessed the capacity of wild-type and opt3-2 to remobilize Fe from one leaf to other leaves using the radio tracer 59Fe. In these experiments, 59Fe was loaded into a mature leaf as Fe2+ at a slightly acidic pH to resemble the apoplastic pH. The addition of ascorbic acid was used to reduce Fe3+ to Fe2+ and maintain it in the reduced form. Figure 7A shows that Fe can be re-mobilized from one leaf to adjacent leaves in wild-type. In contrast,container size for blueberries opt3-2 shows negligible movement of 59Fe between leaves. Figure 7B shows the 59Fe activity in the four leaves adjacent to the leaf where the 59Fe was originally applied. Compared to wild-type, opt3-2 shows a severe reduction in the quantity of 59Fe mobilized from one leaf to the adjacent leaves , suggesting that OPT3 is required for the reallocation of Fe between plant tissues. In fact, opt3-2 plants over-accumulate Fe in mature leaves compared to wild-type, as visualized by Perls’ staining . Interestingly, over-accumulation of Fe in opt3-2 occurs only in mature leaves but not in young leaves . Moreover, accumulation of Fe in opt3-2 is more evident at the base of the trichomes and near the vasculature in the minor veins but not in the main vasculature, suggesting that, in opt3-2, the reallocation of Fe between leaves is impaired, particularly at advanced stages of leaf development . To test whether OPT3 functions as a Fe2+ transporter similarly to IRT1, we expressed OPT3 in a yeast strain deficient in Fe2+ uptake . As previously shown, IRT1 expression in yeast allows the fet3fet4 strain to grow on minimal media without the addition of extra Fe . OPT3 was unable to rescue the fet3fet4 strain , suggesting that, in yeast, OPT3 does not mediate the uptake of Fe2+ like IRT1. Subcellular localization studies, however, show that OPT3–YFP protein fusions do not localize to the plasma membrane in yeast in contrast to in planta .
This mislocalization of OPT3 in yeast precluded further characterization of OPT3 using yeast as a heterologous system. Note that, if fet3fet4 yeast cells were not sufficiently pre-starved of iron, growth of the fet3fet4 mutant was observed, and therefore long-term starvation of yeast was required for these complementation tests. We attempted complementation with different yeast promoters, starting with the strong GAL promoter . Using the phosphoglycerate kinase yeast promoter, OPT3 also did not complement the pre-iron-starved fet3fet4 yeast mutant, consistently with previous studies showing that OPT3 does not complement this yeast mutant . Nevertheless, Fe re-mobilization studies suggest that OPT3 is essential for the remobilization of Fe within plant tissues; whether this transport occurs as Fe2+ or as a Fe-ligand complex remains to be determined. OPT3 is a member of the oligopeptide transporter family and some members of this family have been found to have broad substrate specificity for peptides of different length and amino acid composition . To test whether OPT3 mediates the long-distance transport of GSH in planta, we pursued radio tracer experiments to assess the movement of 35S-GSH from one leaf to adjacent leaves . No differences were found between wild-type and opt3-2, suggesting that OPT3 does not participate in the mobilization of GSH between plant tissues. Interestingly, opt3- 2 rosette leaves from plants exposed to 20 μM CdCl2 and supplemented with 0.5mM GSH accumulated Cd, but no other metals, to wild-type levels. These results suggest that GSH is required for Cd retention in leaves. On the other hand, GSH supplemented to the roots reduced Cd in the leaves of both opt3-2 and wild-type plants likely because GSH trapped Cd in roots of both opt3-2 and wild-type plants . Glutathione has recently been shown to play a critical role in Fe signaling in yeast by stabilizing FeS clusters in the cytosol . In Arabidopsis, GSH is also important to maintain proper homeostasis and crosstalk between Zn and Fe metabolism . To test whether long-distance transport of GSH is important for proper shoot-to-root signaling and homeostasis of trace metals in roots, we measured the constitutive high activity of the root ferric reductase in opt3-2 after foliar application of GSH . In all cases, including the application of foliar GSH, GSH applied to roots, or foliar application of Fe, the activity of the root ferric reductase remained constitutively high in opt3-2. We also tested whether iron applied directly to roots or complexed with GSH, citrate, or nicotianamine is sufficient to repress the high activity of the Fe chelate reductase in opt3-2 roots. Supplemental Figure 6B shows that iron alone , or in complex with GSH, nicotianamine, or citrate, cannot down-regulate the constitutive iron-deficiency response in opt3-2 back to wild-type levels.We have identified an Arabidopsis mutant, opt3-2, that over-accumulates Cd in seeds and roots but, unexpectedly, under-accumulates Cd in leaves . Cadmium distribution throughout the plant is an orchestrated process dictated by root uptake, root-to-shoot translocation through the xylem, and redistribution of Cd from leaves to sink tissues via the phloem. opt3-2 displays constitutive up-regulation of IRT1, a root transporter with broad specificity for heavy metals including Cd . Over-accumulation of Cd, Zn, Fe, and Mn in roots may be explained by the constitutively high expression of IRT1. However, under-accumulation of Cd in leaves and over-accumulation of Cd in seeds, which is different from essential metals , is inconsistent with the high expression of IRT1 . Nutrients, water, and heavy metals are mobilized from leaves into seeds through the phloem . Accumulation of metabolites in sink tissues and under-accumulation in source tissues is best described as an increased redistribution process, likely through the phloem . Notably, of the analyzed metals, only Cd under-accumulates in leaves . These results suggest that, in contrast to the broad specificity of heavy metal uptake at the root level, metal-specific mechanisms mediate the remobilization of heavy metals from leaves to sink tissues. In addition to the altered distribution of heavy metals within the plant leading to over-accumulation of Cd in seeds, opt3-2 also shows hypersensitivity to Cd at the seedling stage . Both the increased accumulation of Cd in seeds and the Cd hypersensitivity of seedling growth are restored to wild-type levels by ectopically expressing OPT3, demonstrating that the altered redistribution of Cd through the plant is the result of the reduced expression of OPT3 in opt3-2 .The opt3-2 mutant displays a constitutive iron-deficiency response in roots, while the leaves properly respond to iron levels as indicated by wild-type levels of ferritin expression , suggesting that the iron status response is mainly disrupted in roots. In plants, the root iron-deficiency response is regulated by local signals within the root and also by unknown systemic signals originating from aerial tissues .