| Phosphate (P) is one of the macronutrients required for plant development and production. PHR1 (Phosphate Starvation Response 1) has been known as the central regulator for Pi-signaling and Pi-homeostasis in plant. The cis-element of PHR1 (PHR1-binding sequence, P1BS) has also been reported. In this work, we investigated the high affinity P1BS structure in rice for the PHR1 homologue OsPHR2 and found the high affinity cis-element (HA-P1BS) which can be used to improve crops with higher Pi uptake ability.Using the method of Yeast-one-hybridization and Electrophoretic mobility shift assay (EMSA), we found that the different affinity of OsPHR2 to the promoters of the genes downstream of OsPHR2, OsIPS1, OsIPS2, OsPT2 and OsPHF1. Among the four promoters, the affinity between the OsIPS2 promoter and OsPHR2 was the most highest, while OsPT2 was the lowest.Further research showed that the variable bases of P1BS and the P1BS-like motif adjacent to P1BS affect the OsPHR2 binding affinity. We analyzed the binding affinity of OsPHR2 to the different substitution of variable bases by the promoter of Pm1 (the P1BS2 was muted in OsIPS2 promoter). Obviously, when the first variant base ’A’changed to ’T’, ’C’or ’G’, and the second variant base ’G’changed to ’C’ ’T’or ’A’, the binding affinities were significantly changed. When ’A’ substituted by ’T’,’C’or ’G’,the OsPHR2 binding affinities were decreased. However there is no obvious difference for the substitution of’G’to’C’or’A’. Also, we observed when the first variant base is’A’and the second variant base is ’T’ (designated this PIBS as A-T-type), the OsPHR2 binding affinity was dramatically increased compared to the other PIBS types. Therefore, we also constructed the PIBS-like motif as A-T-type P1BS and found the two adjacent A-T-type P1BS elements (namely HA-P1BS) results in highest OsPHR2 binding ability.Furthermore, we modified the promoter of OsPHFl (Phosphate Transporter Traffic Facilitator 1), a key factor controlling endoplasmic reticulum-exit (ER-exit) of PT to plasma membrane (PM), with the cis-element of HA-P1BS. The results showed that HA-P1BS significantly increased the expression of OsPHFl, while the tissue expression pattern of OsPHFl driven by HA-P1BS is not changed. Then we developed the transgenic plants of POspHF1-HA-P1BS:PHF1(CDS)(PHF1-HA -P1BS). Physiological tests for two independent lines of PHF1-HA-P1BS were conducted in solution cultures. The transgenic plants with PHF1-HA-P1BS showed significantly higher Pi concentration in both shoots and roots under both high Pi (200 μMPi) and low Pi (10 μM Pi) conditions compared to the wild type plants (Xiushui 134, an japonica high yield variety). The increased Pi-uptake rate was confirmed by 33P-labelld Pi uptake over 24h period and the two PHF1-HA-P1BS lines shown more higher Pi-uptake concentration than wild type under low Pi condition. The pot experiments show that the yield of per plant and total P in grains and shoots of the transgenic plants were increased compared with the wild type under both high and low Pi conditions.In summary, this research discovered the effect of P1BS variable bases and PIBS-like motif for the DNA binding affinity of OsPHR2, a central regulator of Pi-signaling and Pi-homeostasis in rice, and found the HA-P1BS composed of two A-T-type P1BS is the most efficient for the binding of OsPHR2. Besides, the HA-P1BS modified OsPHFl can promote the Pi uptake efficient. This finding provides a new choice for us to cultivate low nutrition tolerance plants by modifying the key regulator promoters. |
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