BnPHT5;1b genes are required for reproductive growth and seed traits
Both BnA09PHT5;1b or BnCnPHT5;1b had strong expression in mature leaves, young leaves and flowers at the reproductive stage (Figure 8a). To evaluate the influence of BnPHT5;1s on reproductive growth, W10 and two BnPHT5;1b DM lines were grown in pots with soil supplemented with 150 mg P/kg soil (normal P supply; NP) and 500 mg P/kg soil (high P supply; HP), respectively (Figure S6 a, b). Under NP conditions, BnPHT5;1b DM plants had shorter stems (plant height) and smaller dry weight than W10 plants, while high P supply improved the growth of BnPHT5;1 DM plants to approximate W10 plants in plant height but not in dry weight (Figure S6c). The total P concentration in stem, leaves and flowers showed that BnPHT5;1 DM plants had larger total P concentration in flowers than in W10 in NP conditions (Figure 8b) and also larger Pi concentrations in the stamen, pistil, petal and sepal (Figure 8c). Under HP conditions,BnPHT5;1b DM plants had larger total P concentration in their flowers while W10 plants had larger total P concentration in the stem (Figure 8d). Therefore, BnPHT5;1b DM plants had distributed more total P to flowers than W10 plants under both NP and HP conditions (Figure S6c, d). A previous study reported that an Arabidopsis mutant lacking vacuolar Pi influx transporter, Atvpt1 Atvpt3 , showed Pi toxicity in floral organs when grown under Pi-sufficient conditions, leading to the inhibition of pollen tube growth and reproductive impairment (Luan et al., 2019). We therefore compared traits related to seed yield between W10 and BnPHT5;1b DM plants. Although there were no significant differences of branch numbers betweenBnPHT5;1b DM plants and W10 plants under NP or HP conditions (Figure S7a, b, d), a decrease in silique numbers and seed yield per plant was observed in BnPHT5;1b DM plants compared to W10 plants under both NP and HP conditions (Figure 8e, f). Therefore, the reduction in seed yield in BnPHT5;1b DM plants could be ascribed to the excessive P/Pi accumulation in floral organs.
Furthermore, we found that seeds from BnPHT5;1b DM plants developed an irregular shape with a concave and convex surface, which differs from the approximately spherical shape of W10 seeds (Figure 9a). The diameter of seeds from BnPHT5;1b DM plants was larger than seeds from W10 plants (Figure 9b), indicating BnPHT5;1b DM had a larger seed size than W10. Indeed, the 1,000-grain weight ofBnPHT5;1b DM seeds was also greater than W10 seeds (Figure 9c). In line with the high P/Pi concentrations in BnPHT5;1b DM flowers (Figure 8b, d), the P concentrations of BnPHT5;1b DM seeds were consistently greater than W10 seeds under both NP and HP conditions (Figure. 9d). However, the germination ratio of seeds from W10 plants was 97.0 ± 3.0%, while the germination ratio of seeds fromBnPHT5;1b DM plants was only 73.0 ± 2.3% and 66.0 ± 3.0% (Figure 9e). These results indicate that BnPHT5;1b genes are required for reproductive growth, seed yield and seed yield related traits in B. napus .