Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

Abstract

ORCID ID: 0000-0001-5953-4144 (T.-J.C.).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 (for isobaric tags for relative and absolute quantitation)-based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

Screening of Maize Genotypes for Drought Tolerance Related Trait Variability

“…Root: shoot ratio increases dramatically under drought conditions suggesting that functional responses to reduced soil moisture primarily occur through increased growth of roots, as has been shown suggested by Quezada and Gianoli (2010). Similarly, Huang et al, (2013) also reported that deficiencies of soil water resulted in high root: shoot ratio. Relatively, more biomass was allocated to the root than to the shoot, and plant allocated more resource to the belowground growth.…”

“…The traits root biomass at bottom and root shoot ratio had increased values under drought 28.463 and 9.388 respectively. Huang et al, (2013) reported that deficiencies of soil water resulted in high root: shoot ratio. Relatively, more biomass was allocated to the root than to the shoot, and plant allocated more resource to the belowground growth.…”

Similarities and Differences in the Acquisition of Fe and P by Dicot Plants

“…The P leaf concentration of the mutant pho1 is very diminished while in the roots it has concentrations very similar to those of the wild cultivar, which suggests that this mutant has a defect in the loading of P into the xylem [100,[102][103][104]. By contrast, the pho2 mutant accumulates up to three times more P in the aerial part than the wild cultivar, while the root maintains similar levels, or even slightly lower levels than the wild cultivar [43,101,105,106]. The pho2 mutant could either prevent the transport of P through the phloem, from the aerial part to the root, or have some problem in the regulation of P concentration in the leaves [107].…”

The Rice CK2 Kinase Regulates Trafficking of Phosphate Transporters in Response to Phosphate Levels

“…Multiple lines of evidence indicate that PT proteins undergo several posttranslational modifications in addition to transcriptional regulation (Bayle et al, 2011;Huang et al, 2013;Lin et al, 2013;Park et al, 2014). One key regulatory step is ER exit of PTs to the PM, which is regulated positively by PHF1 and negatively by phosphorylation.…”