Graduate Thesis Or Dissertation
 

Molecular cloning and characterization of the diageotropica gene in tomato (Lycopersicon esculentum Mill.)

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  • The auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) has a pleiotropic phenotype including a lack of lateral roots and reduced gravitropic response, apical dominance, vascular development, and fruit growth. The dgt mutation reduces the auxin sensitivity of only a subset of auxin responses while levels, metabolism, and transport of auxin appear normal, suggesting that the Dgt gene encodes a component in an auxin-signaling pathway. This dissertation reports isolation and characterization of the Dgt gene. Delineation of three microsyntenic regions in the Arabidopsis genome containing genes homeologous to genetic markers near the Dgt gene allowed isolation of additional ESTs from the corresponding tomato region, significantly reducing the mapping distance to the dgt locus. Further analysis determined that the Dgt gene encodes a cyclophilin (LeCYP1), a previously unidentified component of auxin signaling. Each known dgt allele contains a unique mutation in the coding sequence of LeCyp1. In addition, the wild-type Dgt gene can complement dgt mutant plants. Cyclophilins characteristically have peptidylprolyl cis-trans isomerase (PPIase) activity, but it is unclear whether that activity is necessary for all of their biological functions. Each allelic dgt mutation reduces or nullifies PPIase activity of LeCYP1 fusion proteins in vitro. Immunoblot analysis indicates that all three dgt mutations are null mutations. Phylogenetic comparisons of tomato and Arabidopsis cytosolic-type cyclophilins could not identify any single Arabidopsis member as orthologous to LeCYP1/DGT. Five T-DNA insertion mutants were analyzed to determine if mutations in Arabidopsis cytosolic-type cyclophilins phenocopy the pleiotropic dgt phenotype. Overall seedling growth and morphology appear normal in the mutants, however, their gravitropic response is slow. The lack of exact phenocopy may be due to the redundant nature of Cyp genes in Arabidopsis, which has over twice as many Cyp genes as tomato. In tomato, the cyclophilin inhibitor cyclosporin A (CsA) inhibits auxin-induced adventitious root initiation and expression of two early auxin response genes, LeIAA10 and 11, that are also affected by the dgt mutation. Taken together, these results suggest that the cyclophilin encoded by the Dgt gene plays an important role in auxin signal transduction.
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