Title

Characterization of Polyamine Transporters from Rice and Arabidopsis

Date of Award

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Paul Morris, PhD

Second Advisor

George Bullerjahn, PhD (Committee Member)

Third Advisor

Vipaporn Phuntumart, PhD (Committee Member)

Fourth Advisor

Scott Rogers, PhD (Committee Member)

Fifth Advisor

Emilio Duran, PhD (Committee Member)

Abstract

Uptake and compartmentation of polyamines in plants is crucial for many developmental processes and resistance against biotic and abiotic stresses. Transport of polyamines is a poorly understood component of polyamine homeostasis in plants. In this study, a comparative genomics approach was used to identify and characterize putative polyamine uptake transporters (PUTs) in rice and Arabidopsis thaliana genomes which translocate polyamines. We identified three and four candidate transporter genes in A. thaliana and rice, respectively. These genes are predicted to be membrane proteins with 9-12 transmembrane domains. Functional analysis of PUTs, using yeast complementation assay, demonstrated that all the PUTs tested transported polyamines and paraquat into the cells. Kinetic analysis and competition assays with alternate substrates revealed that all the genes except OsPUT3.2 are high affinity spermidine transporters with Kms ranging from 0.9 µM to 15 µM. Semiquantitative RT-PCR of PUTs showed differential expression at the tissue level suggesting their specialized roles in plant growth and development. Expression analysis using promoter-GUS transgenic lines showed that AtPUT2 is expressed in the vascular tissues of leaves, roots, flower and flower buds indicating that this transporter may be involved in long distance transport of polyamines to the developing plant organs. Strong expression of AtPUT3 in root vascular tissue and lack of expression in the root hairs and root tips suggests that this gene may facilitate the long distance polyamine transport and redistribution within the plant. The subcellular localization of PUTs was determined by fusions with GFP and expression in onion epidermal cells. No convincing results could be obtained for all the PUTs except for OsPUT3.1 which localized to the mitochondria. Further localization studies of rice PUTs in rice protoplasts showed that OsPUT1, OsPUT2 and OsPUT3.2 were localized to the plasma membrane and cytoplasm while OsPUT3.1 localized to the chloroplast. In conclusion, the overlapping substrate specificities along with individual expression patterns of PUTs suggest specific function of each PUT in plant growth and development.