Characterisation of the phytase gene in trifoliate orange (Poncirus trifoliata (L.) Raf.) seedlings

• Trifoliate orange could utilise phytin without microorganisms in P-deficient conditions by promoting phytase and acid phosphatase activity.
• The expressions of the three predicted acid phosphatase genes (PtPAP1, PtPAP2, PtPAP3) of trifoliate orange were up-regulated in roots and leaves with –P solutions.
• The Km values of the recombinant protein (r-PtPAP3) which was expressed in Pichia pastoris were 46.2 μM for phytate and 1631 μM for ρ-nitrophenylphosphate.

Phosphate (P) deficiency decreases the yield and quality of citrus fruits. The P concentration, P uptake, and the expression of three predicted acid phosphatase genes in trifoliate orange seedlings were measured among inorganic phosphate (Pi), organic phosphate (Po), and no phosphate (−P) treatments to assess the ability of Po to improve trifoliate orange P deficiency. The three genes were expressed in Pichia pastoris X-33 to characterise the trifoliate orange phytase gene. Our analysis showed that trifoliate orange could utilise phytin without microorganisms in P-deficient conditions by promoting phytase and acid phosphatase activity; however, Po had a smaller effect on the improvement of trifoliate orange P deficiency than Pi fertiliser. The expression of the three genes in roots and leaves with Pi treatment did not respond to −P solutions, but nearly all three genes were up-regulated in roots or leaves with Po and −P treatment after −P solutions were supplied. Further, the PtPAP3 recombinant protein (r-PtPAP3), which was expressed in P. pastoris, showed phytase activity that was as high as that of cereal phytases. The r-PtPAP3 Km values were 46.2 μM for phytate and 1631 μM for ρ-nitrophenylphosphate. Incubation with Mn2+ increased the specific activity of r-PtPAP3 for phytate by approximately 1.9-fold, and Fe2+ increased it by approximately 1.2-fold. In summary, the function and expression pattern of PtPAP3 allowed a detailed understanding of the significance of phytate metabolism of a woody perennial.