Transcription level of Pht1 transporters is affected by the AM symbiosis
The expression of the Pht1-type Pi transporters was evaluated in mycorrhizal roots of a broad range of plant species and in many cases, modifications in expression were noted. The data are summarized in Table 1. Each Pht1 member is indicated according to the official nomenclature (Karandashov & Bucher 2005), as well as their previously reported names. Here, we refer to the Pi transporters according to this official nomenclature.
For seven plant species analysed, in situ hybridization and promoter::GUS fusion studies indicated that the corresponding mycorrhiza-induced Pht1 genes are predominantly or exclusively expressed in cells containing arbuscules (Rausch et al. 2001; Harrison et al. 2002; Glassop et al. 2005; Nagy et al. 2005; Maeda et al. 2006). Although tomato LYCes;Pht1;1 was initially reported to be induced in cells with arbuscules, it also had an ubiquitous expression level throughout the plant (Rosewarne et al. 1999). The later identification of LYCes;Pht1;4, strongly induced in arbuscules, as well as the detection of two other mycorrhiza-induced Pht1 members in tomato (LYCes;Pht1;3 and 5: Nagy et al. 2005) suggested that the original LYCes;Pht1;1 signal observed around the arbuscule might have resulted from cross-hybridization. In M. truncatula, immunolocalization of MEDtr;Pht1;4 protein revealed that the Pi transporter was detected solely in the plant periarbuscular membrane (Fig. 2; Harrison et al. 2002). The signal was stronger around the fine arbuscular branches and was absent from regions around the hyphal trunk. Furthermore, it could only be detected in developing and mature arbuscules, reinforcing the idea that arbuscules could be the major site of Pi exchange between the two symbionts (Harrison et al. 2002). This expression pattern corroborates previous results suggesting intense active transport on the periarbuscular membrane: plasma membrane H+-ATPases are abundant around fine branches and disappear in decaying arbuscules (Gianinazzi-Pearson et al. 2000). The crucial role played by arbuscules in plant Pi uptake is also reflected by the results of a study on the reduced mycorrhizal colonisation (rmc) tomato mutant (Poulsen et al. 2005). This mutant is characterized by allowing certain AM fungi to develop arbuscules, while halting other AM fungi at various developmental stages of the symbiosis. Results of the study indicated that only those fungi that were able to develop arbuscules triggered the expression of AM-induced LYCes;Pht1;3 and 4, and thus enabled the symbiotic Pi transfer. Therefore, activation of mycorrhiza-induced Pi transporters can be used as a marker for a functional symbiotic Pi uptake pathway.
The heterologous expression of mycorrhiza-inducible Pht1 transporters in yeast has enabled estimates of substrate affinity (Table 1). The mycorrhiza-induced SOLtu;Pht1;3 from potato revealed an apparent Km of 64 µM, whereas the mycorrhiza-specific MEDtrPht1;4 from M. truncatula exhibited a lower affinity for Pi, between 493 and 668 µM (Rausch et al. 2001; Harrison et al. 2002). The biological relevance of these data would be further strengthened if heterologous expression data combined the use of yeast and plant cell culture systems, such as the one developed with tobacco and rice cells (Mitsukawa et al. 1997; Rae et al. 2003).
Formation of the symbiosis and expression of mycorrhiza-induced Pht1 members in arbuscule-containing cells is often accompanied by the down-regulation of other Pht1 transporters, in particular those located at the epidermis (Table 1). To date, down-regulation of Pht1 transporters has been observed in potato (SOLtu;Pht1;1 and 2: Rausch et al. 2001); M. truncatula (MEDtr;Pht1;1 and 2: Liu et al. 1998b); L. japonicus (LOTja;Pht1;1 and 2: Maeda et al. 2006); rice (ORYsa;Pht1;1,2,3,6,9 and 10: Paszkowski et al. 2002); barley (HORvu;Pht1;1 and 2: Glassop et al. 2005) and maize (ZEAma;Pht1;1 and 1;4: Wright et al. 2005). These results reflect the fine balance maintained between fungal and root epidermis uptake pathways. They can also partially explain the results from Smith et al. (2003) showing that even in the absence of any detectable stimulation of the plant Pi content, a significant amount of Pi could be delivered through the fungus. Because the down-regulated Pht1 transporters are also generally responsive to the Pi status of the plant, it is not clear currently whether their down regulation results indirectly from the fungal-induced improvement of the plant Pi status, or from a direct regulation of their expression triggered by the plant in response to the symbiosis. Indeed, in M. truncatula, it was shown that many genes activated by Pi starvation including acid phosphatases and Mt4, are rapidly down-regulated in the AM symbiosis (Burleigh & Harrison 1997, 1998; Liu et al. 1998b, 2003). The Arabidopsis homologue of Mt4 is involved in Pi allocation and its expression may be under the control of a member of the MicroRNA399 family (Shin et al. 2006), which suggests that regulation by MicroRNAs might also operate in the AM symbiosis. A role for the MicroRNA399 in Pi-signaling was very recently demonstrated (Aung et al. 2006; Bari et al. 2006) and in silico analysis, suggests that Pht1 Pi transporters are potential candidates for a regulation by members of the MicroRNA399 family (Jones-Rhoades 2004 #2228; Chiou et al. 2006).
Sequence comparison between characterized fungal Pi transporters, Pht1 members from A. thaliana and AM plant species (Fig. 1) illustrates some interesting features of the Pi transporter family. First, a large majority of mycorrhiza-specific and mycorrhiza-up-regulated Pi transporters cluster into their own subgroups. These subgroups contain only one mycorrhiza ‘non-responsive’ Pht1 member (Zeama;Pht1;5) and do not contain any Arabidopsis Pht1 members. Because Arabidopsis cannot be colonized by AM fungi, this observation suggests that plant species capable of forming mycorrhizas possess specialized types of Pi transporters adapted to the AM symbiosis. Two mycorrhiza-up-regulated Pht1 members (LOTja;Pht1;3 and SOLtu;Pht1;3) do not cluster with this subgroup and cluster with the other Pht1 members, many of which are down-regulated in mycorrhizal roots. Because more than one Pht1 member is induced in mycorrhizal roots of rice, tomato and potato, additional induced transporters will likely be identified in the other plants included in this dendrogram. It will be particularly interesting to see how these new Pht1 members relate to the current groups.
Figure 1. Unrooted dendrogram of plant and fungal phosphate (Pi) transporters. The tree was generated using alignment of full-length protein sequences by ClustalW. Mycorrhiza-specific, up-regulated and down-regulated Pi transporters are highlighted in red, orange and blue, respectively. Transporters and corresponding plant species are: Arabidopsis thaliana, ARAth;Pht1;1 to 9 (Mudge et al. 2002); Tomato, LYCes;Pht1;1,2 and 4 (Liu et al. 1998a; Nagy et al. 2005); potato, SOLtu;Pht1;1 to 4 (Leggewie, Willmitzer & Riesmeier 1997; Rausch et al. 2001; Karandashov et al. 2004; Nagy et al. 2005); Medicago truncatula, MEDtu;Pht1;1, 2 and 4 (Liu et al. 1998b; Harrison et al. 2002); Lotus japonicus, LOTja;Pht1;1 to 3 (Maeda et al. 2006); rice, ORYsa;Pht1;1 to 13 (Paszkowski et al. 2002; Guimil et al. 2005); barley, HORvu;Pht1;1, 2, and 4 to 8 (Rae et al. 2003; Glassop et al. 2005); wheat, TRIae;Pht1;myc (Glassop et al. 2005); maize, ZEAma;Pht1;1to 6 (Wright et al. 2005; Glassop et al. 2005; Nagy et al. 2006). Fungal transporters are Neurospora crassa, PHO5 (Versaw 1995); Saccharomyces cerevisiae, PHO84 (Bun-ya et al. 1991); Glomus versiforme, GvPT (Harrison & van Buuren 1995); Glomus intraradices, GiPT (Maldonado-Mendoza et al. 2001).
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The fact that the family of mycorrhiza-induced Pi transporters includes members from the legumes, solanaceae and grasses, and that down-regulation of Pi transporters in the root epidermis occurs in all these groups, is consistent with the finding that AM symbiosis is a very ancient interaction predating the divergence between mono and dicotyledonous plants (Wolfe et al. 1989; Remy et al. 1994).
Pht2 and other plant transporters
Although the large majority of data regarding Pi transport during AM symbiosis focus on the regulation of the Pht1 type of transporters, it is expected that other components of the Pi transport network play an active part into the reorganization of the Pi fluxes through the plant.
Members of the Pht2 family of Pi transporters have been shown to be involved in the Pi allocation at the whole plant level (Versaw & Harrison 2002), and could therefore be affected during the AM symbiosis. However, all Pht2 members characterized so far (MEDtr;Pht2;1, ARAth;Pht2;1 and SOLtu;Pht2;1) have no, or very moderate responses to Pi level or AM symbiosis (Daram et al. 1999; Zhao et al. 2003; Rausch et al. 2004). Furthermore, the alteration of SOLtuPht2;1 expression did not impact the expression level of a Pht1 Pi transporter expressed in the same cell (SOLtu;Pht1;1: Rausch et al. 2004), raising the question of whether or not these two transporter families are co-regulated at the transcriptional level. Still, a lack of altered transcriptional regulation does not preclude a role for this family in regulating symbiotic Pi allocation. Further assessing the various functions of Pht2 transporters may give rise to new discoveries concerning the reorganization of Pi fluxes within a plant that occurs in response to AM symbiosis. In Arabidopsis, the overexpression of MicroRNA399b down-regulates Pht2;1, and has a differential effect on Pht3;2 and Pht3;3 that is dependant on Pi status (Aung et al. 2006). It will be interesting to see if the MicroRNA399 family plays a role in the regulation of transporters and Pi-signaling pathways in the AM symbiosis.
There is virtually no information regarding the possible participation of the Pht3 type of transporters, or the Pi antiporters/translocators in the reorganization of the Pi transport in response to AM symbiosis. Considering the known effects of AM fungi on plastid and mitochondrial biosynthetic pathways, these transporters are likely involved in channeling Pi transport during AM symbiosis. For example, stimulation of photosynthesis (probably to compensate for the allocation of carbon to the fungus), or carotenoid biosynthesis is observed in many colonized plants, in the shoots and roots, respectively (Jakobsen 1995; Fester et al. 2005). Numerous studies also describe the direct effect of Pi level fluctuations on biosynthetic pathway regulation (Plaxton 2004). The study of Pi translocators is a relatively new area of research, and the involvement of these proteins in the AM association has not yet been investigated. Consequently, the question of their involvement in the AM symbiosis is open, but they are strong candidates for a role in Pi reallocation during this symbiosis.