Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis

Abstract

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8–SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

Mechanistic insights into the regulation of plant phosphate homeostasis by the rice SPX2 – PHR2 complex

“…9a, b). We found that plants carrying mutations in the PHR CC -SPX interface (AtPHR1 L331A ; L352 in OsPHR2 and AtPHR1 L339A ; L360 is OsPHR2) exhibited a growth phenotype similar to the parental phr1 phl1 line, suggesting that these mutations may disrupt the SPX -PHR complex and possibly AtPHR1 oligomerisation 25 . We thus mutated additional polar residues in the PHR CC -SPX complex interface that are located outside the AtPHR1 oligomerisation interface in the previously reported AtPHR1 CC structure 25 .…”

“…Based on this we expressed an engineered SPX 21-202/∆47-59 construct fused to the Cterminus of the macro domain of human histone mH2A1.1 for carrier-driven crystallization 27 . The crystal structure of the mH2A1.1 181-366 tagged SPX2 1-202/∆47-59 /InsP 6 /PHR2 ternary complex was subsequently determined at 3.1 Å resolution, using InsP 6 (phytic acid) as a commercially available surrogate for the bioactive PP-InsPs 22,25 (Fig. 1a, Supplementary Fig.…”

“…4c). It has been previously established that the CC domain of PHRs enables oligomerisation of the transcription factor critical for DNA binding 17,25 . In line with this, EMSA results showed that the DNA binding ability of PHR2 MYB-CC is stronger than PHR MYB (Supplementary Fig.…”

“…10c). Mutation of K325, H328 and R335 at the surface of AtPHR1 CC domain disrupted its interaction with SPX receptors, and led to constitutive Pi starvation responses 25 , whereas the mutation of R318 or R340 produced no effect 25 .…”

Nitrogen and Phosphorus Signaling and Transport During Legume–Rhizobium Symbiosis

“…Phosphate starvation response (PSR) includes both a long-distance, systemic response that involves sensing of cellular Pi level and a local, root tip inhibition response that involves sensing of extracellular Pi availability ( Abel, 2017 ; Puga et al, 2017 ; Ried et al, 2021 ). Cellular Pi sensing relies on the MYB coiled-coil (MYB-CC) transcription factors, PHOSPHATE STARVATION RESPONSE1 (PHR1) and its related proteins PHLs ( Rubio et al, 2001 ) and inositol pyrophosphates (PP-insPs)-binding SPX proteins that exhibit Pi-dependent inhibition of PHR activities ( Puga et al, 2014 ; Wild et al, 2016 ; Ried et al, 2021 ). Under conditions of sufficient Pi, SPX binds to the coiled-coil domain of PHR and prevents the transcription factor from binding the cis -element in the promoter sequences of PSI genes and from activating their expression ( Puga et al, 2014 ; Wang et al, 2014 ; Wild et al, 2016 ).…”