Biology Ph.D. Dissertations

Reimagining How Putrescine Functions as a Signaling Compound: The Essential Role of Synthesis and Compartmentation

Date of Award

2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Paul Morris (Committee Chair)

Second Advisor

Scott Rogers (Committee Member)

Third Advisor

Irina Stakhanova (Other)

Fourth Advisor

Vipaporn Phuntumart (Committee Member)

Fifth Advisor

Zhaohui Xu (Committee Member)

Abstract

Global food insecurity exacerbated by climate change and population is a looming threat. It is imperative that plant scientists identify new strategies to mitigate biotic and abiotic stresses that result in yield reductions. The polyamines, putrescine, spermidine, spermine and thermospermine, are essential metabolites that mediate changes in both developmental and stress responses and thus represent a potential target for metabolic engineering in crop plants. The cationic property of polyamines enables them to interact with other macromolecules and affect cellular processes that include DNA replication, transcription, RNA modification and protein synthesis. Putrescine is the smallest polyamine molecule and is a precursor for the synthesis of the higher polyamines; spermidine, spermine and thermospermine. Multiple pathways mediate putrescine synthesis in plants. The differential expression of rate-limiting enzymes in these pathways suggests that each of these pathways may have different roles. In A. thaliana, arginine decarboxylase 2 and agmatinase are localized to the chloroplast, and function in concert to synthesize putrescine. Localization of the other two pathways for putrescine synthesis has not been established. In this study, transient expression assays of GFP-tagged ornithine decarboxylase from soybeans and rice, showed that both genes were localized to the endoplasmic reticulum. Transient expression assays showed that the rice agmatinase gene is localized to the mitochondria in the mesophyll cells of Nicotiana benthamiana. Similarly, the A. thaliana genes agmatine iminohydrolase and its isoforms (AIH.1, AIH.2, AIH.3, AIH.5) and N-carbamoylputrescine amidohydrolase and its isoforms (NLP1.1, NLP1.2 and NLP1.3) were also localized to the endoplasmic reticulum. Isoform four of agmatine iminohydrolase (AIH.4) exhibits dual localization to the chloroplast and the ER. Thus, two pathways for putrescine synthesis are localized to the endoplasmic reticulum, while a third pathway is localized to the chloroplast. Localization of agmatinase to the mitochondria will also enable the synthesis of putrescine, assuming that mitochondria contain an agmatine shuttle protein. These findings have significant implications for metabolic engineering of putrescine synthesis. Putrescine synthesis is specifically excluded from the cytoplasm. Fluctuations of cytoplasmic putrescine levels are thus better able to function as signaling molecules. The role of specific pathways under different conditions is thus dependent upon the transport of substrates and products in and out of the organelle and enzyme activity. Prior work had shown that NATA1 (N-Acetyl Transferase Activity 1) and ADC1 (Arginine Decarboxylase 1) were localized to the ER and formed a pathway to synthesize acetylputrescine. Colocalization of NATA1 and the enzymes ADC1, AIH and NLP1 to the ER may indicate that when NATA1 is expressed, putrescine is largely converted to acetyl putrescine.

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