Yinghui Ying, Wenhao Yue, Shoudong Wang, Shuai Li, Chuang Wang, Chuanzao Mao, James Whelan and Huixia Shou
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
Phosphate overaccumulator 2 (PHO2) encodes a ubiquitin-conjugating E2 enzyme that is a major negative regulator of the inorganic phosphate (Pi)-starvation response-signaling pathway. A yeast two-hybrid (Y2H) screen in rice using OsPHO2 as bait revealed an interaction between OsPHO2 and two h-type thioredoxins, OsTrxh1 and OsTrxh4 . These interactions were confirmed in vivo using bimolecular fluorescence complementation (BiFC) and by in vitro pull-down assays. Split-ubiquitinY2H analyses and BiFC assays in rice protoplasts confirmed the interaction of OsPHO2 with PHOSPHATE TRANSPORTERTRAFFIC FACILITATOR1 (OsPHF1), and PHOSPHATE1;2 (OsPHO1;2) in the endoplasmic reticulum and Golgi membrane system, where OsPHO2 mediates the degradation of OsPHF1 in both tobacco leaves and rice seedlings. Characterization of rice pho2 complemented lines, transformed with an endogenous genomic OsPHO2 or OsPHO2C445S (a constitutively reduced form) fragment, indicated that OsPHO2C445S restored Pi concentration in rice to statistically significant lower levels compared to native OsPHO2. Moreover, the suppression of OsTrxh1 resulted in slightly higher Pi concentration than that of wild-type leaves. These results demonstrate that OsPHO2 is under redox control by thioredoxins, which fine-tune its activity and link Pi homeostasis with redox balance in rice.
Under Pi sufficient conditions, Pi transporters (PTs) are degraded to prevent excess Pi accumulation. The mechanisms targeting PTs for degradation are not fully elucidated. We found that the Oryza sativa Nitrogen Limitation Adaptation (NLA), OsNLA1 protein, a RING-type E3 ubiquitin-ligase, was predominantly localized in the plasma membrane, and could interact with OsPT2/PT8 and mediate their degradation . Mutation in OsNLA1 (osnla1) led to significant increase of Pi concentration in leaves. Moreover, there was no interaction of OsNLA1 and OsPHO2, suggesting that OsPHO2 was not the partner of OsNLA1 involved in ubiquitin-mediated PT degradation.
 Ying, Y., et al., Two h-Type Thioredoxins Interact with the E2 Ubiquitin Conjugase PHO2 to Fine-Tune Phosphate Homeostasis in Rice. Plant Physiol, 2017. 173(1): p. 812-824.
 Yue, W., et al., OsNLA1, a RING-type ubiquitin ligase, maintains phosphate homeostasis in Oryza sativa via degradation of phosphate transporters. Plant J, 2017.