Plants take up iron from the soil using the IRON-REGULATED TRANSPORTER 1 (IRT1) high-affinity iron transporter at the root surface. Sophisticated regulatory mechanisms allow plants to tightly control the levels of IRT1, ensuring optimal absorption of essential but toxic iron. Here, we demonstrate that overexpression of Arabidopsis thaliana IRT1 leads to constitutive IRT1 protein accumulation, metal overload, and oxidative stress. IRT1 is unexpectedly found in trans-Golgi network/early endosomes of root hair cells, and its levels and localization are unaffected by iron nutrition. Using pharmacological approaches, we show that IRT1 cycles to the plasma membrane to perform iron and metal uptake at the cell surface and is sent to the vacuole for proper turnover. We also prove that IRT1 is monoubiquitinated on several cytosol-exposed residues in vivo and that mutation of two putative monoubiquitination target residues in IRT1 triggers stabilization at the plasma membrane and leads to extreme lethality. Together, these data suggest a model in which monoubiquitin-dependent internalization/sorting and turnover keep the plasma membrane pool of IRT1 low to ensure proper iron uptake and to prevent metal toxicity. More generally, our work demonstrates the existence of monoubiquitin-dependent trafficking to lytic vacuoles in plants and points to proteasome-independent turnover of plasma membrane proteins.ubiquitin | protein dynamic | plant cell biology

Plant growth under low K + availability or salt stress requires tight control of K + and Na + uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K + Transporters), permeable either to K + and Na + or to Na + only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na + transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na + only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na + -K + symport, Na + uniport, or inhibited states, depending on external Na + and K + concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K + and Na + accumulation in monocots.Although it is not clear what levels of Na + are toxic in the plant cell cytosol and actually unacceptable in vivo, the hypothesis that this cation must be excluded from the cytoplasm is widely accepted. The most abundant inorganic cation in the cytosol is K + , in plant as in animal cells. This cation has probably been selected during evolution because it is less chaotropic than Na + (i.e. more compatible with protein structure even at high concentrations; Clarkson and Hanson, 1980). Its selection might also be due to the fact that in primitive cells, which originated in environmental conditions (seawater) where Na + was more abundant than K + , a straightforward process to energize the cell membrane was to accumulate the less abundant cation and to exclude the most abundant one.In the cell, K + plays a role in basic functions, such as regulation of cell membrane polarization, electrical neutralization of anionic groups, and osmoregulation. Concerning the latter function, K + uptake or release is the usual way through which plant cells control their water potential and turgor. Although toxic at high concentrations, Na + can be used as osmoticum and substituted for K and tissue levels have actually been shown to be essential in plant adaptation to salt stress (Greenway and Munns, 1980;Flowers, 1985;Hasegawa...

AKT1, a putative inwardly directed K+ channel of Arabidopsis, restores long-term potassium uptake in a yeast mutant defective in K+ absorption. In this paper, the expression pattern of the gene encoding AKT1 is described. Northern blots indicate that AKT1 transcripts are preferentially accumulated in Arabidopsis roots. Owing to the difficulties in producing large quantities of Arabidopsis roots under hydroponic conditions, experiments were undertaken with Brassica napus, a related species. Potassium starvation experiments on B. napus plants show that changes in the K+ status of the organs do not modify AKT1 mRNA accumulation. Western blot analysis of B. napus proteins confirms the presence of AKT1 at the root plasma membrane. Tissue-specific expression directed by the Arabidopsis AKT1 gene promoter was investigated by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing an AKT1-GUS gene fusion. As determined by fluorimetric and histochemical tests, the AKT1 promoter directs preferential expression in the peripheral cell layers of root mature regions. The discrete activity found in leaves relates to leaf primordia and to small groups of cells, hydathodes, found on toothed margins of the Arabidopsis leaf lamina. These data are discussed with regard to a possible role of AKT1 in K+ nutrition of plants.