| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 104 === MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHOSPHATE2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative membrane proteomic method was employed to search for components downstream of PHO2. Among 7491 proteins identified from Arabidopsis thaliana roots, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family, PHO1 and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2. We showed that PHO2 interacts with PHO1 and PHT1 in the post-endoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. We further demonstrated that PHO2 mediates the degradation of PHT1 and PHO1 proteins. Our study uncovers a mechanism by which PHO2 modulates Pi acquisition and translocation by posttranslational regulating PHO1 and PHT1 proteins in the endomembrane compartments, and also provides a database of the membrane proteome in root biology research.
Plant vacuoles serve as the primary intracellular organelle for Pi storage. Vacuolar Pi pool plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. Several lines of evidence indicate that the SPX-MFS proteins, designated as PHOSPHATE TRANSPORTER 5 family (PHT5) function as vacuolar Pi transporters. Based on 31P nuclear magnetic resonance spectroscopy analysis, Arabidopsis pht5;1 loss-of-function mutants accumulate less Pi and exhibit a lower vacuolar-to-cytoplasmic Pi ratio than wild-type plants. Conversely, overexpression of PHT5 leads to stunt growth, massive Pi sequestration into vacuoles and altered regulation of Pi starvation-responsive genes. Our results demonstrate a group of Pi transporters in vacuolar membranes that regulate cytoplasmic Pi homeostasis and are required for plant fitness and growth.
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