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 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 level 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, the expression of AAT Rhg1 proteins mutated 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 transport and homeostasis of amino acids.