Get connected to the fungal network for improved transfer of nitrogen: the role of ZmAMT3;1 in ammonium transport in maize-arbuscular mycorrhizal symbiosis

When building your house and considering options for water supply, you can decide to build your own pipes all the way from the spring to your house, but most of us would find it easier to pay a company to get connected to an existing distribution network. So do plants! Most plants have evolved the strategy of plugging into the arbuscular mycorrhizal fungus (AMF) network to get the nutrients they need. Such symbioses give the plant access to a much larger soil volume at a low construction cost. The system relies on the trade of organic carbon from the plant, mostly lipids, for mineral nutrients from the fungus, mainly phosphorus and nitrogen (N; Smith and Smith, 2012). While the detail of phosphorus nutrition in this two-body system is well established, less is known about the mycorrhizal N-uptake. AMFs take up and assimilate N from the soil, and then release inorganic N, namely nitrate and ammonium (${\text{NH}}_4^{+}$/NH₃) into the interfacial apoplast between the root cell and its arbuscule. Wang et al. (2020) recently shed light on symbiotic nitrate uptake in rice, elucidating the presence of a nitrate transporter, leaving the question of ammonium uptake. Now, Jing Hui and colleagues (Hui et al., 2022) reveal a dedicated ammonium transporter in maize (Zea mays).

First identified via transcriptomic analysis, the ammonium transporter gene ZmAMT3;1 was found to be highly upregulated in AMF-inoculated roots. Its expression was determined to be root-specific and AMF-dependent while not being dependent on the AMF species. In addition, expression levels of ZmAMT3;1 over time correlated with increasing AMF colonization after inoculation, similar to the mycorrhizal root-specific maize phosphate transporter gene ZmPht1;6. ZmAMT3;1 expression was detected specifically in the arbuscule-containing cortical root cells using in situ hybridization with the ZmAMT3;1 or using a GUS reporter. Expression of a ZmAMT3;1-GFP fusion construct under the native promoter showed exclusive localization in arbuscular cortical cells, mostly at the periphery of arbuscular branches. Taken together, the results suggest that ZmAMT3;1 is present only in the peri-arbuscular membrane.

To validate the function of ZmAMT3;1, a heterologous-expression approach was used. The expression of ZmAMT3;1 in an ammonium uptake-defective yeast strain restored growth with ammonium as sole N source. Kinetic properties assessed using an ¹⁵N-labeled ammonium influx assay in yeast indicated that ZmAMT3;1 is a high-affinity ammonium transporter. Similar experiments conducted with heterologous expression in oocytes showed that the transporter was also active. In both systems, activity of the transporter was independent of external pH ranging from 3.5 to 7 pointing toward ${\text{NH}}_4^{+}$ as the substrate rather than NH₃. However, electrophysiological assays in oocytes did not show any inward current as would be expected with an influx of ${\text{NH}}_4^{+}$. Therefore, ZmAMT3;1 most likely recruits ${\text{NH}}_4^{+}$ but transfers uncharged NH₃ across the membrane.

As the mycorrhizal N-uptake pathway coexists with direct root N-uptake from the soil, the authors next investigated the expression of two root epidermis-expressed ammonium transporters, ZmAMT1;1a and ZmAMT1;3, which are major components of direct root N uptake. Although they found similar transcript abundance after mycorrhizal inoculation, protein abundance was significantly reduced, and their C-terminus showed typical phosphorylation associated with post-translational downregulation of the transporter activity. Overall, activation of the mycorrhizal N-uptake pathway appears to be associated with repression of the direct root N-uptake pathway.

Finally, to assess the importance of ZmAMT3;1 in vivo, RNAi transgenic lines were generated in maize. Inhibition of ZmAMT3;1 did not affect mycorrhizal colonization. However, using ¹⁵N-labelled soil fraction, accessible only to the hyphae, ZmAMT3;1 was shown to be essential for mycorrhizal N-uptake in maize. An experimental pot setup allowing a hypha-exclusive soil portion showed a decrease of total N-uptake in the RNAi lines leading to the conclusion that ZmAMT3;1 accounted for about 70% of the mycorrhizal-dependent N-uptake. Subsequent field experiments showed that silencing of ZmAMT3;1 mainly affected N acquisition after flowering, when ZmAMT3;1-mediated mycorrhizal N-uptake accounted for about 30%.

Overall, ZmAMT3;1 was shown to be an AMF-inducible transporter critical for the transfer of ammonium across the peri-arbuscular membrane providing molecular understanding of the mycorrhizal N-uptake pathway (see Figure). Although plant-AMF symbioses are widespread, their benefits for plant N nutrition in field and environmental conditions are debated (Makarov, 2019). This study provides evidence for a beneficial contribution of AMF to N-uptake, opening the opportunity to address the challenge of improving N-acquisition efficiency in crops using AMF symbiosis. Overall, AMF can function as an extended supply system for both nitrogen and phosphorus, just as a single utility company can provide you with water and electricity!

Figure.

Arbuscular mycorrhiza-dependent N uptake in maize roots relies on ZmAMT3;1. Direct root uptake and mycorrhizal uptake coexist in AMF-symbiotic plants. Absorption of nitrogen by the fungus is metabolized into arginine (Arg), which can be catabolized into ammonium. The latter is released in the interfacial apoplast of symbiont by an unknown mechanism and absorbed by the root cell thanks to a mycorrhizal specific ammonium transporter ZmAMT3;1 at the peri-arbuscular membrane. The mycorrhizal N-uptake pathway inhibits the direct root N-uptake pathway. The symbiotic interface is also known to host nitrate and phosphate uptake from the fungus. Adapted from Hui et al. (2022), Figure 10.