Overexpression of phyA and appA genes improves soil organic phosphorus utilisation and seed phytase activity in Brassica napus

Abstract

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg(-1) seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.

Figure 1. Construction of transgenic plants overexpressing A. niger phyA or E. coli appA.

A and B: Schematic representation of binary vector pBI121-phyA (A) and pBI121-appA (B) for B. napus plant transformation. Phytase genes (A. niger phyA and E. coli appA) under the control of the CaMV 35S promoter and nos-terminator were modified for extracellular secretion by inclusion of an extracellular targeting sequence from the carrot extensin (ex) gene. The construct has a selectable marker for the NPT?? gene. C and D: Southern blotting analysis of ex::phyA (C) and ex::appA (D) transgenic lines, using phyA and appA probes, respectively. Genomic DNA was digested by HindIII and EcoRI. C, M, Marker; WT, wild-type canola; 1–4,T₃ transgenic plants of P3 line; 5–6, T₃ transgenic plants of P11 line; D, 1–4, T₃ transgenic plants of a18 line. E and F: Northern blotting analysis of phyA (E) and appA (F) expression in roots of transgenic lines using phyA and appA probe, respectively.

Figure 2. Phytase activity in leaf (A) and root (B) extracts of transgenic lines when grown in different P conditions.

HP: plants were grown in high P (250 µM KH₂PO₄) nutrient solution for 18 days; LP: plants were grown in low P (5 µΜ KH₂PO₄) nutrient solution for 18 days; Phy-P: plants were grown in 250 µM P in the form of phytate for 18 days. Each column is the mean of four replicates with SD. Different letters represent significant differences at the p<0.05 level.

Figure 3. Phytase activity (A) and acid phosphatase activity (B) exuded from roots of transgenic lines expressing ex::phyA/appA and WT.

Plants were grown in different P treatment solutions. Each column is the mean of four replicates with SD. Different letters represent significant differences at the p<0.05 level.

Figure 4. Growth response of transgenic plants and WT plants in sand culture.

A: Phenotype of transgenic lines (P3, P11 and a18) and WT plants grown for 60 d in quartz sand culture with different P sources. B: Leaf number and size of transgenic lines and WT plants when grown in quartz sand with phytate as the sole P source. The leaves arranged from left to right ranged from the oldest to youngest according to the growth order in the plant.

Figure 5. Phytase activity (A) and phytic acid content (B) in seeds of transgenic T₃ lines and WT.

Each column is the mean of four replicates with SD. Different letters represent significant differences at the p<0.05 level. The seeds were harvested from the field by self-cross. n.d., means not detectable.