AbstractThree plant pathways for the synthesis of putrescine have been described to date. These are the synthesis of putrescine from ornithine, by ornithine decarboxylase (ODC); and the synthesis of putrescine from arginine by arginine decarboxylase, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (NLP1); or arginine decarboxylase and agmatinase. Several enzymes associated with putrescine synthesis have yet to be localized. Here we showed that ODC in soybeans and rice was localized to the ER. In rice, agmatinase is localized to the mitochondria. In A. thaliana there are five isoforms of AIH and three isoforms of NLP1. Stable GFP-tagged transformants of the longest isoforms of AIH and NLP1 showed that both proteins were localized to the ER in leaves and roots of A. thaliana. Four of the isoforms of AIH and all of the isoforms of NLP1 were localized to the ER. However, AIH1.4 was localized to both the ER and the chloroplast. Combining these results with other published data, reveal that putrescine synthesis is excluded from the cytoplasm and is spatially localized to the chloroplast, ER and likely the mitochondria. Synthesis of putrescine in the ER may facilitate cell to cell transport via plasmodesmata, or secretion via vesicles. Differential expression of these pathways may enable putrescine-mediated activation of hormone-responsive genes.