Medicago truncatula NPF1.7: Structure-Function Assessment and Potential as a Phytohormone Transporter
Authors:   , Yu Yao Chuan 
Published: January 2025

In Medicago truncatula, the MtNPF1.7 transporter has been shown to be essential for root morphology and nodulation development. The allelic MtNPF1.7 mutants, Mtnip-1 (A497V), Mtnip-3 (E171K), and Mtlatd (W341STOP), show altered lateral root growth and compromised legume-rhizobium symbiosis. To assess the role of a series of distinct amino acids in the transporter's function, in silico structural predictions were combined with in planta complementation of the severely defective Mtnip-1 mutant plants. The findings support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. The results also question the existence of a putative TMH4-TMH10 salt bridge, which may not form in MtNPF1.7. Results reveal that a motif conserved among MFS proteins, Motif A, is essential for function. Hypothetically, the Motif A participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. The mutated valine (A497V) in Mtnip-1 may interfere with the lateral helix. Mutating a residue (L253) on the lateral helix with reduced side chain restored Mtnip-1 function. The predicted residue (Q351) for substrate binding is not essential for protein function. To probe the possibility that MtNPF1.7 transports auxin, two heterologous assay systems were attempted. The first was a Xenopus laevis oocyte assay. However, MtNPF1.7 expressed in oocytes failed to show substrate transport, which may due to low expression levels of proteins on the membrane or may be caused by other factors. Second, yeast (Saccharomyces cerevisiae) strains expressing MtNPF1.7 were constructed. They showed an increased flux of radiolabeled IAA and differential susceptibility to 5-fluoroindole-3-acetic acid (F-IAA), a toxic IAA-like compound. These results suggested that MtNPF1.7 may function as an auxin transporter in yeast. Unexpectedly, the Mtnip-1 (A497V) and Mtnip-3 (E171K) proteins when expressed in yeast also showed influx of F-IAA transport in yeast, suggesting the auxin transport remains functional in the mutants, potentially contradicting other results suggesting Mtnip-1 is non-functional. To address the possible role of MtNPF1.7 in auxin responses, the auxin reporter DR5-GUS was used and mutant plants were subjected to gravitropism tests. The auxin reporter DR5::GUS displayed different expression in Mtnip-1 mutant in comparison with DR5::GUS expression in wild-type. In addition, the spatial expression of pMtNPF1.7-GUS partly co-localized with the expression of DR5:GUS in roots and nodules. The Mtnip-1 plants showed altered gravitropic responses to gravistimulus compared to wild-type and Mtnip-3 plants. The results suggest that MtNPF1.7, recognized as a nitrate transporter, may be able to transport auxin and may participate in auxin distribution during M. truncatula root and nodule development. These research findings helps elucidate the complex function of plant NPF transporters.