Isolation and characterization of two peptide transporter cDNAs from Arabidopsis thaliana

Author

Wei Song

Date of Award

12-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Daniel Roberts, David Hacker, Jeffrey Becker, Beth Mullin

Abstract

The transport of small peptides of 2 to 5 residues across the plasma membrane, a phenomenon shown to occur in a range of prokaryotes and eukaryotes, is mediated by specific membrane proteins in an energy-dependent manner. Peptide transport has been found to be involved in a number of biological processes such as nutrition, antibiotic and toxin absorption. Peptide transport systems have been well studied in a wide variety of organisms including bacteria, fungi, and animals, but there are only a handful of reports on their occurrence in plants. However, several lines of evidence have demonstrated that peptide transport not only exists in plants, but may also participate in important physiological processes in plants such as nutrition. The major goal of my project was to isolate and characterize the genes required for peptide transport in plants, and further elucidate the physiological role of these identified peptide transporters with regard to plant growth and development. This document describes the isolation of two peptide transporter cDNAs, AtPTR2-A and AtPTR2-B, from an Arabidopsis thaliana cDNA library by functional complementation of a yeast peptide transport mutant deficient in ptr2,/em>, a yeast peptide transporter gene. Transfer of AtPTR2-A or AtPTR2-B to the yeast ptr2 mutant restored the ability to grow on di- and tripeptides but not peptides of four residues or longer. Transformants expressing AtPTR2-A or AtPTR2-B were also sensitive to toxic di- and tripeptides. Kinetic studies indicated that AtPTR2-A and AtPTR2-B have K_m, values of 47 &um;M and 14 &um;M, respectively, with V_max values of 0.061 and 0.013 nmoles/mg cell dry weight (sec)^-1 respectively, when dileucine was used as a substrate. DNA sequence analysis showed that AtPTR2-A encodes a putative 68 kD protein (611 aa) and AtPTR2-B encodes a putative 64 kD protein (585 aa). Hydropathy analysis indicated that both proteins are highly hydrophobic with twelve putative transmembrane domains. A search of the protein sequence data base reveals a high degree of similarity of AtPTR2-A and AtPTR2-B to the yeast peptide transporter Ptr2p, the A. thaliana nitrate transporter AtCHL1, the rabbit intestinal peptide transporter PepT1, the Candida albicans peptide transporter CaPtr2p, and Lactococcus lactis peptide transporter DtpT. Comparison of these protein sequences to a number of other transport proteins suggested that these transporters comprise a new family of membrane transport proteins, called the PTR (Peptide TRansport) family. Northern blot analysis showed that AtPTR2-B was highly expressed in roots, leaves, germinating seeds, stems, flowers, and siliques. AtPTR2-A mRNA could not be detected by Northern blot analysis, but was detectable in roots after reverse transcription and PGR amplification. Transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B or AtPTR2-A. Southern blot analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were regenerated. Southern blot analysis showed that these lines had 1 or 2 T-DNA copies integrated into their genome. This was supported by the segregation data following kanamycin resistance encoded on the T-DNA. Northern blot data showed that the endogenous AtPTR2-B mRNA levels was significantly reduced in transgenic leaves and flowers. Consistent with this reduction, all antisense lines and one sense transgenic line exhibited significant phenotypic changes including late flowering, arrested seed development, and increased lateral root and root hair formation. These phenotypic changes may reflect a nutritional deficiency due to reduced peptide transport mediated hy AtPTR2-B, suggesting that AtPTR2-B may play a general role in plant nutrition.