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- Materials and Methods
- Supporting Information
Arbuscular mycorrhizal (AM) symbioses are ancient and widespread, occurring in both natural and agronomic plant ecosystems. The symbioses are often considered to be mutualistic. The AM fungi are obligate symbionts dependent upon carbon (C) supplied by the plant host and, in exchange, provide the plant with mineral nutrients from the soil, in particular phosphate (Pi). However, plant responses to AM colonization are highly variable. While some plant species demonstrate considerable increases in growth and Pi uptake upon AM colonization, there are also species that demonstrate negligible or even negative growth responses. Such species have been identified in a wide variety of plant families, including both natural and agricultural species (Tawaraya, 2003). Here we call them ‘nonresponsive’ to denote lack of positive response in terms of plant phosphorus (P) and growth, while recognizing that other responses to colonization may exist. The cereals wheat (Triticum aestivum) and barley (Hordeum vulgare) are considered to be nonresponsive. Nevertheless, colonization under field conditions has been consistently reported (Jensen & Jakobsen, 1980; Graham & Abbott, 2000; Aliasgharzadeh et al., 2001; Li, 2005; Grace, 2008). It has been proposed that agricultural crops represent a great potential for improvement of growth and yield responses and/or reduction of inputs such as P fertilizer via manipulation of AM symbioses (Grace et al., 2008; Sawers et al., 2008). However, a better understanding of the interaction, particularly in nonresponsive plants, is necessary to realize this potential.
Growth depressions resulting from AM colonization are conventionally attributed to C loss to the fungal symbiont with no subsequent gain in fitness from increased access to plant nutrients (Stribley et al., 1980; Graham & Abbott, 2000). Indeed, fungal C demand has been estimated to be as high as 15–20% of plant photosynthates (Jakobsen & Rosendahl, 1990; Wright et al., 1998). However, such ‘demand’ will only be a net C drain if there are no compensatory processes, such as decreased exudation of organic C, or reduced root to shoot weight ratios in AM plants (Johnson et al., 1997). Recently, Li et al. (2008) questioned the validity of applying the ‘C demand’ analysis universally in all nonresponsive AM interactions. In wheat colonized by either of two AM fungi, an equivalent growth depression was observed despite distinct differences in the C demand, as measured by per cent AM colonization in roots and hyphal length density in soil. Similar observations have been reported previously (Hetrick et al., 1992), but have thus far received little attention. Li et al. (2008) hypothesized that regulation of Pi uptake rather than C demand may explain such growth depressions. They postulated that the suppression of plant Pi uptake pathways with no net gain via fungal uptake pathways will result in lower overall Pi uptake and hence plant growth depressions.
Arbuscular mycorrhizal plants have two pathways for uptake of Pi from the soil solution. Direct Pi uptake occurs via the root epidermis and root hairs, whereas in the AM pathway Pi is taken up by external AM hyphae and transported to intracellular symbiotic interfaces within colonized root cortical cells. Previous assumptions that plant and fungal uptake pathways were additive in their contribution to plant nutrient accumulation led to the conclusion that the AM Pi uptake pathway is nonfunctional in AM plants, which do not accumulate additional P compared with nonmycorrhizal (NM) controls (i.e. nonresponsive plants). However, this simplistic notion has been largely overturned by methodologies that enable quantification of the actual contribution of the AM pathway. Using 33P labelling and compartmented pot systems, Smith et al. (2003, 2004) calculated a 100% contribution of Glomus intraradices to both flax (Linum usitatissimum), which showed a positive mycorrhizal growth response, and tomato (Solanum lycopersicum; formerly Lycopersicon esculentum), which showed a growth depression. Using a similar calculation for wheat, the AM Pi uptake pathway contributed 80% of plant P, but there was no difference in growth or P content between AM and NM plants (Li et al., 2006). While demonstrating that the AM uptake pathway is functional in nonresponsive plants, these data also indicate that P inflow via the direct uptake pathway can be reduced.
The plant transporters involved in Pi uptake via both direct and AM pathways are Pi : H+ symporters of the Pht1 family (Bucher et al., 2001; Smith, 2002; Bucher, 2007). In the direct uptake pathway genes encoding Pht1 transporters are expressed primarily in the root epidermis and root hairs (Leggewie et al., 1997; Daram et al., 1998; Liu et al., 1998a; Chiou et al., 2001; Mudge et al., 2002). Schunmann et al. (2004) showed that HvPT1 (HORvu;Pht1;1) and HvPT2 (HORvu;Pht1;2) of barley are strongly and specifically expressed in trichoblast cells of the root epidermis and in root vascular tissue. This expression pattern suggests a role in both Pi uptake from the soil and loading into the vascular system. Studies on the uptake activity of HvPT1 demonstrated high-affinity transport activity with a Km of 9.06 µm (Rae et al., 2003), consistent with a role in the accumulation of Pi from the very low concentrations in soil solution (typically < 10 µm) (Bieleski, 1973). Genes of the Pht1 family involved in the AM uptake pathway are upregulated in AM roots. Their products have been localized to the periarbuscular membrane of colonized cortical cells where they are involved in the acquisition of Pi released by the fungus at this symbiotic interface (Rausch et al., 2001; Harrison et al., 2002; Karandashov & Bucher, 2005). Such AM-inducible Pht1 transporter genes have also been observed in cereals and include HvPT8 (HORvu;Pht1;8) in barley (Paszkowski et al., 2002; Glassop et al., 2005, 2007; Guimil et al., 2005; Nagy et al., 2006).
The Pi transporter genes encoding proteins involved in the direct uptake pathway are downregulated under high P conditions and are responsive to the overall P status of the plant (Muchhal et al., 1996; Leggewie et al., 1997; Smith et al., 1997; Liu et al., 1998a,b; Rausch & Bucher, 2002; Schunmann et al., 2004). Concurrent changes in transcript abundance and protein levels indicate that regulation of these transporters is primarily transcriptional (Muchhal & Raghothama, 1999; Chiou et al., 2001). In AM plants, downregulation of Pi transporter gene expression has also been observed in response to colonization. In positively-responsive Medicago truncatula, the expression of the root epidermal Pi transporter genes, MtPT1 (MEDtr;Pht1;1) and MtPT2 (MEDtr;Pht1;2), decreased with increasing AM colonization (Liu et al., 1998b). Downregulation of MtPT2 varied with AM fungal species and it was suggested that this response is primarily a function of the improved P status of the plant (Burleigh et al., 2002). However, down-regulation of Pi transporter genes has also been observed in nonresponsive AM hosts, which do not show increases in tissue P concentrations. In nonresponsive rice (Oryza sativa), six of ten root-expressed Pi transporter genes were downregulated on AM colonization (Paszkowski et al., 2002). In barley, HvPT1 and HvPT2 were down-regulated in AM roots despite similar shoot and root P concentrations (Glassop et al., 2005). In contrast to data from responsive Medicago, these results suggest an AM-specific signalling pathway involved in downregulation of root-expressed Pi transporter genes that is independent of the P response pathways in the plant.
Overall, the data for transporter gene expression are consistent with the finding that P inflow via the direct pathway is reduced in nonresponsive AM plants where the AM Pi uptake pathway is functioning. In the present study, we used a combined physiological and molecular approach to further investigate the link between the contribution of the AM Pi uptake pathway and the expression of Pi transporter genes in the nonresponsive AM host, barley, in association with two AM fungi that differ significantly in colonization of the plant.