MurA-catalyzed synthesis of 5-enolpyruvylshikimate-3-phosphate confers glyphosate tolerance in bryophytes

Glyphosate is a broad-spectrum herbicide that kills most vascular plant weeds but is ineffective against many bryophytes. Glyphosate competitively inhibits the enolpyruvyl transferase enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS catalyzes the production of 5-enolpyruvylshikimate-3-phosphate (EPSP)-an intermediate in the shikimate pathway-from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) substrates. Here, we show that <i>Marchantia polymorpha</i> mutants with loss-of-function mutations in a closely related enolpyruvyl transferase, Mp<i>MurA,</i> were more sensitive to glyphosate than wild-type controls. Overexpression of Mp<i>MurA</i> in <i>M. polymorpha</i> increased glyphosate tolerance, and heterologous expression of the <i>M. polymorpha</i> MurA enzyme in <i>Arabidopsis thaliana</i> conferred glyphosate resistance. Furthermore, we demonstrate that MpMurA catalyzes the production of EPSP from S3P and PEP substrates. These data demonstrate that MpMurA contributes to glyphosate tolerance in <i>M. polymorpha</i>. We speculate that the existence of two independent mechanisms for EPSP synthesis-one canonical and EPSPS-dependent, and the other MurA-dependent-may account for glyphosate tolerance in bryophytes. Alternatively, MurA in bryophytes may bind glyphosate, thereby leaving more unbound EPSPS enzymes available, allowing aromatic amino acid biosynthesis to continue.