Preferential ammonium: nitrate ratio of blueberry is regulated by nitrogen transport and reduction systems

Blueberry (Vaccinium spp., Vc) is inefficient in uptake and utilizing a single nitrogen (N) form (ammonium or nitrate), and prefers the presence of both N forms; however, its preferential ammonium: nitrate ratios and the underlying molecular mechanism remain elusive. To reveal the mechanism of blueberry preference in the composition of N forms, blueberries had grown over three months in pure coconut bran irrigated with modified Hoagland nutrient solution containing ammonium N (NH₄⁺-N) alone or nitrate N (NO₃⁻-N) alone at two pHs (5.0 and 7.5), and NH₄⁺-N:NO₃⁻-N ratios of 4:1, 2:1 and 1:1 at pH 5.0. After three months, the physiological responses and the expression of key genes involved in N metabolism were analyzed. Afterwards, the genes were then genetically transformed into Arabidopsis thaliana for functional verification. The results showed that (i) plants grew better at pH 5.0 than pH 7.5; (ii) at pH 5.0, when NH₄⁺-N:NO₃⁻-N ratio was applied at 2:1, plant growth was at the best performance (i.e., 11% higher in the crown width compared to the control(nil-N applied). In the presence of both NH₄⁺ and NO₃⁻, the transcriptional levels of VcAMT3, VcNRT1.5, VcNRT2, VcNR and VcNiR were significantly upregulated. The highest value appeared in the 2:1 of NH₄⁺-N:NO₃⁻-N ratio where, VcAMT3 and VcNRT2 were raised by 4 and 5 times, respectively. Hence, the results suggested that the mixed N forms at 2:1 NH₄⁺-N:NO₃⁻-N ratio at pH 5.0 induced the expression of the related gene, which may have increased root absorption and transportation of NH₄⁺-N and NO₃⁻-N, resulting in the better plant growth; and (iii) under NH₄⁺-N alone treatments, the transgenic VcNiR Arabidopsis thaliana plants were the only survival; under NO₃⁻-N alone treatments, compared to the wild-type plants, the transgenic VcAMT3 Arabidopsis thaliana had 11 and 2 times higher in the plant height and leaf disc size. The results showed that VcAMT3, VcNRT2, VcNiR genes could improve the nitrogen absorption ability of Arabidopsis thaliana plants. Our study improved the understanding of the underlying molecular mechanism of N speciation preference of blueberry, and the findings can be a great guidance for better N fertilizer management to promote blueberry productivity.