Soybean Cyst Nematode-Resistant Protein AAT_Rhg1 Affects Amino Acid Homeostasis and Betalain Accumulation

Amino acid transporters play crucial roles in plant nitrogen metabolism but also in defense responses. AAT_Rhg1, an apparent amino acid transporter encoded by Glyma.18g022400 (Rhg1-GmAAT) at the soybean Rhg1 locus, contributes to resistance to soybean cyst nematode (SCN), although the in planta function of AAT_Rhg1 remains elusive. In this study, we discovered that overexpression of Rhg1-GmAAT in soybean roots enhances the betalain pigment synthesis driven by a RUBY transgene cassette, potentially through its transporter activity affecting tyrosine levels and amino acid homeostasis. Silencing Rhg1-GmAAT also moderately increased betalain accumulation, while co-overexpression of Rhg1-GmAAT and GmRBOHG (encoding an AAT_Rhg1-interacting NADPH oxidase) blocked the betalain phenotype, indicating a complex role of AAT_Rhg1 in regulating cellular metabolism. Soybean AAT_Rhg1 did not show a betalain accumulation phenotype when co-overexpressed with RUBY in Nicotiana benthamiana leaves, suggesting that soybean AAT_Rhg1 functions differently in N. benthamiana. In soybean, expression of AAT_Rhg1 proteins with mutations at conserved residues D122A or Y268L mitigated or enhanced the betalain phenotypes, respectively, suggesting that these residues are important for AAT_Rhg1 function. This study advances our understanding of AAT_Rhg1 while presenting a novel strategy for enhancing betalain biosynthesis by modulating the transport and homeostasis of amino acids.