RORγ Inhibitor manufacturer phosphate starvation (9, 10, 19, 20). To determine whether or not PHR1 may be involved in AtFer1 gene expression in planta, we isolated a PHR1 loss-of-function mutant. This mutant, named phr1-3, was obtained in the Salk (line SALK_067629) and was previously characterized (19). Accumulation of AtFER1, 3, andVOLUME 288 Number 31 AUGUST two,22672 JOURNAL OF BIOLOGICAL CHEMISTRYPhosphate Starvation Directly Regulates Iron Homeostasiscould be associated to an alteration on the response of this gene to an iron excess in this genetic background. To challenge this hypothesis, the potential of AtFer1 gene to be up-regulated in response to iron overload was assayed in the phr1-3 background (Fig. 2B). Plants were grown for 19 days in a manage medium and treated for three h with 500 M Fe-citrate. This remedy was previously shown to de-repress the expression in the AtFer1 gene and results in a robust increase in κ Opioid Receptor/KOR Inhibitor site abundance of its transcript (4, 5, 23). In phr1-3 mutant, AtFer1 mRNA transcript abundance was strongly increased, as well as the level reached was close for the a single observed in wild kind plants, indicating that the impact of PHR1 on AtFer1 gene expression is not linked to a defect in the gene response to iron overload beneath phosphate starvation. These results show that phosphate starvation leads to an increase of AtFer1 mRNA abundance, and that this response is PHR1 dependent. By contrast, expression of other ferritin genes will not be altered by phosphate deficiency, which is constant with all the lack of P1BS sequence in their promoter. Furthermore, the PHR1-dependent Pi-deficiency response of AtFer1 is unrelated to an alteration in the iron responsiveness of this gene. PHR1 and PHL1 Regulation of AtFer1 Expression Is Independent on the Plant Iron Status–As observed in Fig. 2, PHR1 regulates only partially the AtFer1 response to phosphate starvation. Considering that gel shift experiments (Fig. 1C) showed that PHL1 was also able to bind to Element two inside the AtFer1 promoter region, we hypothesized that the residual amount of AtFer1 transcript observed in the phr1-3 mutant in response to phosphate starvation could be as a result of PHL1 activity. To challenge this hypothesis, a PHL1 loss of function mutant, phl1-2 (SALK_079505), was isolated and crossed with phr1-3 mutant plants. AtFer1 mRNA abundance was monitored during a time course soon after phosphate starvation in wild type, phr1-3, phl1-2, and within the phr1 phl1 double mutant. Plants had been grown hydroponically for 10 days inside a comprehensive medium and transferred to a phosphate-free medium. Shoots and roots were collected 3 to 9 days right after transfer towards the Pi medium. AtIPS1 was used as a constructive manage on the efficiency of phosphate starvation (data not shown). In leaves (Fig. 3A) of both wild variety and phl1-2 plants, AtFer1 mRNA abundance was low through the five initial days of phosphate starvation, and was strongly increased (by 15-fold) following 7 and 9 days. In phr1-3 leaves, an increase of AtFer1 transcript abundance was still observed, but to a decrease extent than in wild type leaves. This result is constant with those presented in Fig. 2A. AtFer1 mRNA increase in abundance was totally abolished in the leaves from the phr1 phl1 double mutant (Fig. 3A). In roots (Fig. 3B), the profile of AtFer1 mRNA abundance was reminiscent of those observed in leaves for each wild type and phl1-2 plants, nonetheless having a higher increase in abundance (by 25-fold right after 7 days). In both phr1-3 and phr1 phl1 mutant plants, the AtFer1 response to phosphate star.