OsbHLH6 interacts with OsSPX4 and regulates the phosphate-starvation response in rice.

Low soil phosphorus (P) availability is a major limitation for crop production. The molecular mechanisms underlying plant response and adaptation to phosphate (Pi) deficiency are unclear. OsbHLH6 (hereafter bHLH6), an uncharacterized Pi starvation-response gene encoding a basic helix-loop-helix protein, was identified by yeast two-hybrid screening using the phosphate response-repressor OsSPX4 (hereafter SPX4) as bait. bHLH6 is expressed in shoots and roots, with its expression significantly induced in shoots by Pi deficiency. bHLH6 overexpression lines showed Pi accumulation and enhanced Pi-starvation responses including upregulation of Pi-starvation induced (PSI) genes and longer root hairs. bhlh6 mutant showed no significant phenotype variation at seedling stage. Pull down assay indicated that bHLH6 had higher binding affinity with SPX4 compared to OsPHR2, therefore, bHLH6 competitively inhibited the interaction of SPX4 and OsPHR2. SPX4 overexpression rescued the Pi accumulation caused by bHLH6 overexpression under high- and low-P conditions. Moreover, overexpression of bHLH6 in spx4 background did not affect the Pi content of spx4 under high- and low-P conditions. bhlh6 / spx4 double mutant showed lower shoot Pi concentration and transcript abundance of OsPT3 and OsPT10 compared with spx4 mutant under high-P conditions. RNA sequencing results indicated that bHLH6 OV and spx4 share many differentially expressed Pi-responsive genes. Therefore, bHLH6 is an important regulator for Pi signaling and homeostasis which antagonizes SPX4. This knowledge helps elucidate the molecular regulation of plant adaptation to Pi deficiency and will promote efforts toward the creation of low Pi-tolerant crops.