Doctoral Dissertations

Serry Koh

Date of Award

12-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Committee Members

Jeffrey M. Becker, Barry Bruce, Albrech von Arnim, Neil Quigley

Abstract

AtPTR2 is a peptide transporter capable of transporting di- and tripeptides in Arabidopsis thaliana. Previous studies showed that antisense expression of AtPTR2 cDNA resulted in an arrest of seed development (Song et al., 1997). In order to gain more insight into the possible function of AtPTR2, a highly specific polyclonal antibody was raised to AtPTR2 and used for immunolocalization studies. Western blot analysis localized AtPTR2 to a plasma membrane enriched fraction isolated from 3 week-old seedlings. Consistent with this observation, immunogold labeling, using transmission electron microscopy, localized AtPTR2 to the plasma membrane in all tissues examined. Immunolocalization with anti-AtPTR2 antibody, using silver enhanced light microscopy, showed that the protein was present in the root, stem and developing seed. AtPTR2 was found exclusively in the maternal tissue of the developing seed. Reciprocal crosses between transgenic plants expressing AtPTR2 cDNA in the antisense orientation and wild-type A. thaliana showed a pronounced maternal effect on seed development. These data support our hypothesis that AtPTR2 is a plasma membrane localized peptide transporter that plays a crucial developmental role in the maternal tissue of the developing seed. More recently, eight more di- and tripeptide transporter orthologues (AtPTR2C to AtPTR2J) were identified in the database search. In addition to the PTR family, eight oligopeptide transporter orthologues (AtOPT1 to AtOPT8) were identified by a search of the Arabidopsis thaliana sequences in the database. These proteins show significant sequence similarity to Candida albicans CaOpt1p and oligopeptide transporters identified in Schizosaccharomyces pombe and Saccharomyces cerevisiae (Isp4p and Opt1p). Hydrophilicity plots of the AtOPTs suggest that they are integral membrane proteins with 12-14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct family from the PTR family and form a discrete subfamily when compared to the fungal OPTs. The identification of multiple peptide transporters in Arabidopsis suggests that they may play different functional roles. This idea was supported by the fact that the AtOPTs exhibited a distinct, tissue-specific expression pattern. The cDNA encoding each of the AtOPTs was cloned into a yeast vector under the control of a constitutive promoter. When expressed in S. cerevisiae, five out of the seven AtOPT proteins tested conferred the ability to uptake tetra- and pentapeptides. To determine the physiological roles of AtOPTs in plants, T-DNA knockout mutants of AtOPT2 and AtOPT3 genes were identified using a PCR-based screening method. Single T-DNA insertion mutant lines in both AtOPT2 (G5-1 line) and AtOPT3 (N4 line) were identified by Southern blot analysis and DNA sequencing. The AtOPT2::T-DNA mutant (G5-1 line) showed a defect in leaf pattern formation resulting in a narrow leaf. This leaf phenotype cosegregated with Kanamycin resistance encoded on the T-DNA. The AtOPT3::T-DNA N4 line showed a seed abortion phenotype resulting in a low seed number per silique. Seed clearing experiments revealed the absence of developing embryos in the albino seeds produced by the N4 line. Segregation analysis of the N4 line showed a 2:1 ratio (KmR:KmS) consistent with the idea that the aborted seeds are homozygous recessive for the AtOPT3::T-DNA mutation.

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