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Arbuscular mycorrhiza (AM) is a mutualistic endosymbiotic association between fungi of the ancient phylum Glomeromycota and most vascular land plant species (Remy et al., 1994). More than 80% of terrestrial plant species are known to be able to form AM symbiosis (Denison & Kiers, 2011). This mutualism improves the plant's efficiency for uptake of mineral nutrients, particularly phosphates, and in return fungi obtain organic carbon from the plant to complete the fungal life cycle (Smith & Read, 2008). To form AM symbiosis, the two symbionts undergo a series of developmental transitions that enable the fungus to enter the root cortex and establish highly dichotomously branched arbuscules in which phosphate and other nutrients are delivered to the root (Bucher, 2007). During arbuscule development, a plant-derived membrane, the peri-arbuscular membrane (PAM) develops around the branching hypha and separates the fungus from the plant cell cytoplasm. Phosphate transport proteins essential for symbiotic inorganic phosphate (Pi) transfer to the plant cell reside in this membrane (Harrison et al., 2002). Symbiotic phosphate transport in AM plants is mediated by specific phosphate transporters (Karandashov & Bucher, 2005). Phosphorus (P), as an essential mineral nutrient, plays a key role in AM symbiosis. Because of phosphate's low mobility and thus its availability in soil (Ai et al., 2009), plants adopt a series of adaptive strategies to increase the acquisition of poorly available Pi. One of them is the formation of symbiosis with AM fungi.
Plant Pht1 transporters belong to the phosphate: H+ symporter (PHS) family within the major facilitator superfamily (MFS; Pao et al., 1998). So far, Pht1 transporters in the direct uptake pathway occurring through the epidemal cells have been observed in potato (StPT1 and StPT2; Rausch et al., 2001); Medicago truncatula (MtPT1 and MtPT2; Liu et al., 1998b); rice (OsPT1-10; Paszkowski et al., 2002); and barley (HvPT1 and HvPT2; Rae et al., 2003). AtPHR1 and OsPHR2 are key regulators in the Pi signalling pathway (Jia et al., 2011), and overexpression of the two genes results in excessive accumulation of Pi in shoots, and upregulation of the Pht1 genes under Pi-sufficient conditions (Liu et al., 2010). Currently, only a few of these Pi transporters have been functionally characterized. The double pht1;1 and pht1;4 mutant from Arabidopsis thaliana exhibits a significant decrease in Pi uptake and shoot accumulation under both Pi-sufficient and Pi-deficient conditions, indicating compensatory effects between the two transporters (Shin et al., 2004), and AtPht1;5 plays a critical role in mobilizing Pi from source to sink organs in accordance with the P status of the plant (Nagarajan et al., 2011). The fourth Pht1 member functionally characterized so far, AtPht1;9 transporter, mediates root Pi acquisition under Pi-deprived conditions, contributing to the overall plant Pi status (Remy et al., 2012).
Several mycorrhiza-specific Pht1 members have been identified in M. truncatula (MtPT4), rice (OsPT11), potato (StPT4 and StPT5) and tomato (SlPT4) (Harrison et al., 2002; Paszkowski et al., 2002; Nagy et al., 2005; Chen et al., 2007; Xu et al., 2007). Tissue localization studies revealed that the mycorrhiza-induced Pht1 genes are exclusively expressed in arbuscule-containing cells (Harrison et al., 2002). The spatial expression pattern of these genes suggests that a cell autonomous signal involved in arbuscule development, activates expression of these genes (Gomez-Ariza et al., 2009). Lysophosphatidylcholine (LPC) may be one of these signals, as it was shown to trigger mycorrhiza specific Pi transporter gene expression in potato and tomato (Drissner et al., 2007). Functional analysis of these Pi transporter promoters revealed that P1BS and MYCS elements are required to confer the AM-inducible PiT gene expression (Chen et al., 2011). MtPT4 is essential for symbiotic Pi transport and the development of AM symbiosis (Javot et al., 2007b). The mycorrhiza-inducible Pi transporters StPT3, StPT4 and StPT5 from potato, exhibit functional redundancy (Nagy et al., 2005). Knockdown of LjPT3 exhibits a decrease in Glomus mosseae arbuscules and necrotic root nodules (Maeda et al., 2006). However, the functions of other mycorrhiza-specific Pi transporters have not been described.
Here, we report on the characterization of two novel mycorrhiza-specific Pi transporters, AsPT1 and AsPT4, and four other Pht1 family members from Astragalus sinicus, designated as AsPT2, AsPT3, AsPT5 and AsPT6, respectively. Both AsPT1 and AsPT4 play critical roles during the development of AM symbiosis, but AsPT4 alone is required for symbiotic Pi uptake.