Glutamine synthetase GLN1;5 and ACT domain repeat ACR1 functionally interact at the seed level in Arabidopsis...

GLN1;5 is one of the five Arabidopsis thaliana glutamine synthetase (GS) cytosolic isoenzymes, almost exclusively expressed in seeds, where it plays a critical role in nitrogen reassimilation during seed maturation and germination. ACT Domain Repeat (ACR) proteins are believed to act as nutrient sensors, although their precise functions in plants remain unclear. The eFP browser expression analysis shown that one ACR isogene, AtACR1, entirely overlaps with AtGLN1;5 expression, suggesting a possible interaction between these proteins. This study aimed to elucidate the functional relationship between the two proteins, using an integrated approach combining phenotypic, biochemical, and molecular analyses. The similarity in phenotypes of the two mutants, acr1 and gln1;5 highlights the crucial role of both proteins in nutrient utilization during germination and supports their potential interaction. Furthermore, in acr1 seeds, GS activity was reduced despite increased protein levels and gene expression, especially of the GLN1;5. Pull-down assays using recombinant proteins and seed extracts from wild-type, acr1, and gln1;5 mutants confirmed a specific interaction between ACR1 and GLN1;5. In addition, recombinant ACR1 protein enhances GS activity in wild-type but not in gln1;5 mutant seed extracts, indicating a regulatory role of ACR1 in GLN1;5 activation. Hormonal treatment of wild-type seed revealed antagonistic regulation of ACR1 and GLN1;5 genes by abscisic and gibberellic acid during germination, further supporting a role for ACR1 in modulating GS activity during this process. By using complementary and alternative approaches, our findings establish ACR1 as a key regulator of glutamine synthetase in seeds, with implications for normal seedling development.