Biology Ph.D. Dissertations

Title

A new perspective on polyamine biosynthesis and transport in arabidopsis thaliana

Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Paul Morris (Advisor)

Second Advisor

Joshua Blakeslee (Committee Member)

Third Advisor

Scott Rogers (Committee Member)

Fourth Advisor

Vipaporn Phuntumart (Committee Member)

Fifth Advisor

Dryw Dworsky (Other)

Abstract

Polyamines are low molecular weight aliphatic organic compounds that are present in all living organisms and are essential for cell viability. Polyamine homeostasis in cells is maintained by their biosynthesis and transport. To better understand how polyamines regulate plant developmental response pathways, we studied the effects of expression of spermidine preferential polyamine uptake transporters, PUT5:OsPUT1 and CaMv:OsPUT1 in A. thaliana. Overexpression of these transporters resulted in plants with larger leaves, thicker stems and a delay in flowering time. In contrast, the homozygous mutant of AtPUT5, had smaller leaves, thinner stems and flowered earlier than wildtype plants. HPLC analysis of plant polyamines suggested that these phenotypes are correlated with elevated levels of spermidine and spermidine conjugates in the leaves of transgenic plants.

These results intrigued us to discover new polyamine biosynthesis pathways that could contribute to polyamine homeostasis in plants under different environmental conditions. There are two known biosynthetic pathways to synthesize putrescine. The first pathway utilizes ornithine decardoxylase (ODC) to convert ornithine to putrescine. The second pathway utilizes arginine decarboxylase (ADC) to convert arginine to agmatine and two additional enzymes, agmatine deiminase (AI) and N-carbamoyl putrescine aminohydrolase (NLP1) to complete this pathway. Here we show the existence of a novel putrescine biosynthesis pathway in A. thaliana. Confocal microscopy experiments show that both the enzymes ADC2 and ARGAH2 are localized to the chloroplast in A. thaliana and soybeans. In addition, transformation of a yeast strain deficient in polyamine biosynthesis with ADC2 from A. thaliana and arginases from A. thaliana and soybean (AtARGAH1, AtARGAH2 or GmARGAH) complemented the WT phenotype. Further evidence for this pathway comes from HPLC analysis of in vitro assays of the enzymes, AtADC2 and AtARGAH2. This analysis confirmed that these two enzymes function in concert to convert arginine to agmatine and then to putrescine.

Putrescine synthesized in the chloroplast via this new pathway should be transported out of the chloroplast in to other plant tissues. Therefore, we identified and characterized a potential polyamine exchanger, AtBAT1. Heterologous expression of AtBAT1 in an E. coli mutant deficient in polyamine antiporters and measurement of the uptake of radiolabeled substrate into inside-out membrane vesicles prepared from E. coli cells expressing the protein revealed that this protein is a proton-mediated high efficiency transporter of putrescine and spermidine. Competition assays showed that BAT1 also functions as a high affinity GABA and arginine transporter. Since BAT1 was originally identified as a broad substrate amino acid transporter, we used competition assays to show that the uptake of spermidine is inhibited by mM levels of alanine, glutamate and agmatine. Transient expression of BAT1.1-GFP in the leaves of Nicotiana benthamiana showed that BAT1 was localized to the ER in cells surrounding leaf veins and the chloroplast membrane of mesophyll cells. Since the chloroplast serves as a site for the synthesis of arginine, agmatine, GABA, and putrescine, BAT1 can regulate the export of these metabolites from the organelle.

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