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Three plant pathways for the synthesis of putrescine have been described to date. These are the synthesis of putrescine from ornithine, by ornithine decarboxylase (ODC); the synthesis of putrescine from arginine by arginine decarboxylase, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (NLP1); and arginine decarboxylase and agmatinase. To address how these pathways are organized in plants, we have used transient expression analysis of these genes in the leaves of Nicotiana benthamiana. Brassicas do not have ODC, but the single ODC gene from rice and one of the soybean genes, were localized to the ER. Transient expression of the rice agmatinase gene showed that it was 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, but in tissues with chloroplasts, the localization was concentrated to lamellae adjacent to chloroplasts. Transient expression analyses showed that four of the isoforms of AIH and all of the isoforms of NLP1 were localized to the ER. However, AIH.4 was localized to 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Two biosynthetic routes are known for putrescine, an essential plant metabolite. Ornithine decarboxylase (ODC) converts ornithine directly to putrescine, while a second route for putrescine biosynthesis utilizes arginine decarboxylase (ADC) to convert arginine to agmatine, and two additional enzymes, agmatine iminohydrolase (AIH) and N-carbamoyl putrescine aminohydrolase (NLP1) to complete this pathway. Here we show that plants can use ADC and arginase/agmatinase (ARGAH) as a third route for putrescine synthesis. Transformation of Arabidopsis thaliana ADC2, and any of the arginases from A. thaliana (ARGAH1, or ARGHA2) or the soybean gene Glyma.03g028000 (GmARGAH) into a yeast strain deficient in ODC, fully complemented the mutant phenotype. In vitro assays using purified recombinant enzymes of AtADC1 and AtARGAH2 were used to show that these enzymes can function in concert to convert arginine to agmatine and putrescine. Transient expression analysis of the soybean genes (Glyma.06g007500, ADC; Glyma.03g028000 GmARGAH) and the A. thaliana ADC2 and ARGAH genes in leaves of Nicotiana benthamiana, showed that these proteins are localized to the chloroplast. Experimental support for this pathway also comes from the fact that expression of AtARGAH, but not AtAIH or AtNLP1, is co-regulated with AtADC2 in response to drought, oxidative stress, wounding, and methyl jasmonate treatments. Based on the high affinity of ARGAH2 for agmatine, its co-localization with ADC2, and typically low arginine levels in many plant tissues, we propose that these two enzymes can be major contributors to putrescine synthesis in many A. thaliana stress responses., (Published by Elsevier B.V.)