Advances in the Characterization of Entities Mediating Root K Uptake

Although the characterization in heterologous systems together with gene expression patterns strongly suggested that many of the aforementioned entities participated in root K+ uptake, a clear demonstration was still pending. It was necessary to demonstrate the transport activity of such systems in vivo, in their native environment. Furthermore, a subcellular localization compatible with K+ uptake (plasma membrane localization) was also required.

The first successful study on this topic demonstrated that an Arabidopsis T-DNA knock-out mutant in AKT1, atakt1-1, had reduced plant growth in the presence of 2 mM NH4+ when external K+ was below 1 mM (Hirsch et al. 1998). These results suggested that K+ channels could also mediate K+ uptake in the high-affinity range of concentrations. Further studies showed the presence of two components of K+ uptake: an NH4+-insensitive AKT1-mediated component and an NH4+-sensitive component (Spalding et al. 1999). A later study showed that AKT1 fused to

GFP localized to the plasma membrane in tobacco mesophyll protoplasts (Hosy et al. 2005). AtAKT1 function is regulated by the AtKC1 subunit, whose gene is strongly expressed in roots (Reintanz et al. 2002) and upregulated by salt stress in the shoot (Pilot et al. 2003). When AtKC1 is coexpressed together with AtAKT1 in heterologous systems, a shift in the activation threshold toward more negative values is observed (Duby et al. 2008; Geiger et al. 2009), probably preventing AKT1 from mediating K+ efflux. Recently, it has been shown that AtKC1 forms a ternary complex with AtAKT1 and a syntaxin (SYP121) that mediates K+ uptake in Arabidopsis roots (Honsbein et al. 2009). When expressed in Xenopus oocytes, the characteristics observed in this ternary complex are more similar to those observed in native Arabidopsis roots than those recorded without the syntaxin (Honsbein et al. 2009), indicating that in planta, the functional unit is the ternary complex.

As for HAK transporters, experiments performed with T-DNA insertion mutants in Arabidopsis demonstrated that root high-affinity K+ uptake was impaired in the athak5-1, athak5-2, and athak5-3 mutants which lacked a functional AtHAK5, (Gierth et al. 2005; Rubio et al. 2008). Moreover, in the athak5 plants, NH4+ did not inhibit Rb+ uptake, while this cation did so in WT plants, indicating that AtHAK5 mediates the NH4+-sensitive high-affinity K+ uptake in plants, in agreement with the sensitivity to NH4+ that AtHAK5 shows when expressed in yeast (Rubio et al. 2000). Recently, it has been shown that adult athak5-3 plants display lower plant biomass due to reduced K+ uptake when they are grown for several weeks at 10 mM K+ (Nieves-Cordones et al. 2010), evidencing that this transporter supports growth at very low-external K+ concentrations. These results are in agreement with another study performed in agarose plates in which AtHAK5 mutants seedlings exhibited reduced root growth when grown below 10 mM K+ (Qi et al. 2008). Moreover, in this study, it was also reported that AtHAK5 fused to an epitope of the human influenza virus hemaglutinin protein (HA epitope) localizes to the plasma membrane.

Fig. 4.1 Molecular entities participating in K+ uptake in Arabidopsis roots. The graphic shows the external K+ concentrations at which the high- and the low-affinity K+ uptake systems operate. In the high-affinity range, AtHAK5 is the only system mediating K+ uptake when the external concentration is below 10 mM. At higher concentrations, AtHAK5, AtAKTl, and AtCHX13 may be the main systems participating in this process. In the low-affinity range of K+ concentrations, AtAKTl, AtCHX13, unidentified members of the CNGC or unknown systems could contribute to K+ uptake. Dashed horizontal lines depict the range of external K+ concentrations in which the system just above plays its important role in root K+ uptake

Fig. 4.1 Molecular entities participating in K+ uptake in Arabidopsis roots. The graphic shows the external K+ concentrations at which the high- and the low-affinity K+ uptake systems operate. In the high-affinity range, AtHAK5 is the only system mediating K+ uptake when the external concentration is below 10 mM. At higher concentrations, AtHAK5, AtAKTl, and AtCHX13 may be the main systems participating in this process. In the low-affinity range of K+ concentrations, AtAKTl, AtCHX13, unidentified members of the CNGC or unknown systems could contribute to K+ uptake. Dashed horizontal lines depict the range of external K+ concentrations in which the system just above plays its important role in root K+ uptake

As stated above, AtAKTl also contributes to K+ uptake in the high-affinity range of concentrations. Recent studies with single athak5, ataktl and double athak5, atakt1 mutants in combination with NH4+ and Ba2+ that inhibit AtHAK5 and AtAKTl, respectively, have allowed the establishment of the relative contributions of each of these two systems to K+ uptake from diluted solutions (Rubio et al. 2010) . AtHAK5 is the only system-mediating K+ uptake at concentrations below 10 mM. Between 10 and 50 mM K+, AtHAK5 and AtAKTl have been demonstrated to be the only systems contributing to K+ uptake. Above 50 mM K+. both systems are thought to act, and at concentrations higher than 200 mM, the contribution AtHAK5 decreases and the only system operating is probably AtAKTl, although the contribution of other unknown systems cannot be ruled out (Fig. 4.1).

Recent research has demonstrated that members of the HKT transporter/channel family mediate important Na+-tolerance mechanisms in plants mainly by improving K+/Na+ homeostasis. Previous work based on the identification of a quantitative trait locus (QTL) determining salt tolerance showed that Knal in Triticum aestivum controls the selectivity of Na+ and K+ transport to shoots, resulting in a high K+:Na+ ratio in leaves (Dubcovsky et al. 1996; Gorham et al. 1987, 1990; Luo et al. 1996). Other loci identified by QTL analyses in durum wheat, Nax1 and Nax2, also contributed to salt tolerance (Lindsay et al. 2004; Munns et al. 2003). Furthermore, the presence of Nax1 and Nax2 was shown to enhance K+ accumulation in leaf blades and sheaths, leading to the model that Nax1 and Nax2 mediate K+-loading into the xylem (James et al. 2006). These QTLs are attributable to polymorphisms in copies of wheat HKT genes, TmHKT1;4-A1 and TmHKT1;4-A2 (also named TmHKT7-A1 and -A2) for Nax1 and TmHKT1;5 and TaHKT1;5 for Nax2 and Kna1 (Byrt et al. 2007; Huang et al. 2006) + Independent analysis of another QTL in rice, named SKC1 (shoot K+ content), resulted in an identical model for the function of the rice gene OsHKT1;5 (Ren 2005). The SKC1 QTL was due to point mutations in OsHKT1;5 that replace several amino acid residues in the salttolerant cultivar NonaBokra (Ren 2005) .

Studies performed in different null mutant types of the class I HKT transporter AtHKT1 has shed light into the mechanisms by which this entity controls Na+/K+ homeostasis under salt stress. According to these studies, AtHKT1 may be involved in Na+ recirculation in plants by operating in Na+ loading in the phloem (Berthomieu et al. 2003) or removal of Na+ from the xylem preventing the accumulation of Na+ in the leaves (Davenport et al. 2007; Horie et al. 2006; Sunarpi et al. 2005). In agreement with this model, AtHKT1 overexpression in the root pericycle improves salt tolerance (Moller et al. 2009).

Concerning K+ homeostasis, mutations in OsHKT1;5 and AtHKT1 have also been found to reduce K+ and enhance Na+ accumulation in shoots during salt exposure, contributing to enhanced salinity stress (Ren 2005; Sunarpi et al. 2005). Interestingly, the disruption of AtHKT1 in mutants of the salt overly sensitive (SOS) pathway prevented the K+-deficiency symptoms observed in sos mutants when grown at low K+ concentrations and improved cellular K+/Na+ ratios when compared with single sos mutants under saline conditions (Rus et al. 2001, 2004).

All the results described above refer to HKT transporters belonging to the class I of this family. They have been shown to mediate Na+ transport and the effect upon K+ nutrition may be indirect. On the other hand, some members of the Class II of HKT transporters can operate as Na+-K+ sym-porters under some conditions (Haro et al. 2005; Jabnoune et al. 2009; Rubio et al. 1995), and a contribution to K+ uptake may be expected. However, mutations in the OsHKT2;1 gene do not have a strong impact on high-affinity K+ (Rb+) uptake into intact rice roots (Horie et al. 2007). Many of the presently characterized HKT class two genes are also induced by K+ starvation, including those in wheat, barley, and rice (Garciadeblas et al. 2003; Horie et al. 2001; Wang et al. 1998) ; Therefore, in addition to mediating K+ uptake, this system mediates Na+ uptake, allowing Na+ to act as a substitute nutrient for K+ in K+-starved rice plants under moderate Na+ concentrations (Horie et al. 2007) , supporting the long-standing hypothesis that Na+ may substitute for K+ when this nutrient is scarce (Flowers et al. 1983; Mengel and Kirkby 1982).

Regarding CHX transporters, it has been shown that disruption of AtCHX17 led to lower root K+ concentrations under saline and K+-deprivation conditions, although a subcellular localization and a functional characterization for AtCHX17 remain to be assessed (Cellier et al. 2004) ; On the other hand, AtCHX13 was localized to the plasma membrane and AtCHX13 knock-out and over-expressing mutant plants showed impairment and enhancement of K+ uptake, respectively (Zhao et al. 2008). All these results suggested that this transporter may be involved in K+ uptake in planta.

With respect to CNGCs, AtCNGC10 is targeted to the plasma membrane, transports both K+ and Na+ and partially rescues Arabidopsis akt1 mutant plants (Kaplan et al. 2007). Results obtained in antisense lines of this channel, which displayed lower expression than WT plants, indicated a role in Na+/K+ homeostasis in roots by providing a pathway for Na+ influx and K+ efflux at the root/soil interface (Guo et al. 2008). Similar results were obtained for AtCNGC3. A null mutation in this channel altered both short-term Na+ influx and K+ uptake at high-external K+ conditions, suggesting an alternate role in nonselec-tive monovalent cation uptake at the plasma membrane level (Gobert et al. 2006).

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