Study on the absorption and accumulation of nitrate and the expression of nitrate transporter genes in pineapple

Abstract:【Objective】Reducing nitrogen use and improving nitrogen use efficiency are two of the central concerns in pineapple cultivation. Nitrate transporters (NRT) play important roles in the absorption, transportation, redistribution and signaling of nitrate and other nutrients, but their roles in nitrate absorption and redistribution in pineapple remain unclear.【Methods】Two experiments were carried out. The first was application of 30 mmol·L-1 NH4NO3 to pineapple plantlet, and changes of nitrate contents and expressions of 40 nitrate nitrogen transporter genes in pineapple roots, stems and leaves were analyzed every 2 h from 10:00 to 18:00; The second was application of six treatments (30 mmol·L-1 NH4NO3, 60 mmol·L-1 NH4NO3, MS nutrient solution containing 60 mmol·L-1 NH4NO3, nitrogen-deficient MS nutrient solution, MS nutrient solution and water) to pineapple plantlet, and changes of nitrate contents and expressions of 40 nitrogen transporter genes in leaves were analyzed in 3rd d, 6th d, 9th d and 26th d after treatment, and root, stem and leaf weights were analyzed 60th d after treatment.【Results】The nitrate contents of pineapple roots, stems and leaves all showed higher values at both 10:00 and 16:00; 2 h after nitrate application (30 mmol·L^-1 NH4NO3), the nitrate content in roots increased significantly; nitrate application also increased the nitrate content in leaves at 18:00, while it decreased the nitrate content in stems at 14:00 and 18:00 as well as that in roots at 16:00. Twenty- five NRT genes were highly expressed in roots. Among them, the highest expression levels across all sampling time points appeared at 12:00 for 2 genes that were postponed (AcNRT1.14) to 14:00 or weakened (AcNRT1.12) after nitrate application; expression levels of 13 genes peaked at 14:00 that were delayed to 14:00 (AcNRT1.25, AcNRT1.17, AcNRT1.20, AcNRT1.8, AcNRT1.19, AcNRT1.21, AcNRT1.32, AcNRT1.24, AcNRT1.23, AcNRT1.35, AcNRT1.22, AcNRT3.1) or to 16:00 (AcNRT1.42) after nitrate application; expression levels of AcNRT1.31 peaked at 18:00, which were advanced to 14:00 after nitrate application; for other genes, the times when the expression peaked were not altered but the expression level was enhanced (AcNRT1.9, AcNRT1.17, AcNRT1.18, AcNRT1.11, AcNRT1.33) or weakened (AcNRT2.3, AcNRT1.35, AcNRT1.38, AcNRT1.12) by nitrate application. Four NRT genes were highly expressed in stems. Among them, expression levels of AcNRT1.7 peaked at 14:00 and were enhanced till 18:00 by nitrate application; expression levels of AcNRT1.17 peaked at 12:00 and 18:00, and both peaks were enhanced by nitrate application; expression levels of AcNRT1.5 and AcNRT1.37 peaked at 18:00 and were weakened by nitrate application. Twelve NRT genes were highly expressed in leaves. After nitrate application, AcNRT1.31, AcNRT1.41, AcNRT1.2, AcNRT2.1 and AcNRT1.36 were up-regulated at 12:00; AcNRT1.34 was up-regulated at 14:00; AcNRT1.27 and AcNRT1.10 were up- regulated at 18:00; AcNRT1.44 were up- regulated at both 12:00 and 18:00; AcNRT1.38 were up-regulated at both 12:00 and 14:00; AcNRT1.26 were up-regulated at 12:00, 16:00 and 18:00; AcNRT1.4 was down-regulated at 18:00. The six different nitrogen fertilization treatments had different effects on the growth of pineapple plantlet. The MS nutrient solution treatment had the largest weight gain, followed by the pure nitrogen treatment (30 and 60 mmol·L^-1 NH4NO3). The plant weight gain of treatment with nitrogen- deficient MS nutrient solution and water was the least, but the former promoted root growth. At 3rd d after treatments, except for the treatment of water (control), the nitrate content in leaves of pineapple plantlet with all other treatments increased. At 6th d after treatments, except for the treatment of MS nutrient solution containing 60 mmol·L^-1 NH4NO3, the nitrate content in leaves of plants with all other treatments decreased, indicating that the assimilation of nitrate was enhanced. At 9th d after treatment, except for the treatments of pure nitrogen (30 and 60 mmol·L^-1 NH4NO3), the nitrate content in leaves of plants with all other treatments decreased, indicating that plants treated with pure nitrogen-initiated accumulation of nitrate. At 26th d after the treatment, the nitrate content of leaves with all treatments decreased significantly, indicating that the leaves were undergoing a nitrogen- deficient status. Three days after treatment, only a few genes were up-regulated by the treatments (e.g. AcNRT 1.2, AcNRT 1.43 and AcNRT 1.3 by treatment of nitrogen- deficient MS nutrient solution; AcNRT 1.18, AcNRT1.5 and AcNRT 1.37 by water control), and there was no common up-regulated gene among all nitrogen-containing treatments; most genes reached their peak expression at 6th day in leaves with water control (AcNRT 1.17, AcNRT 1.42, AcNRT 1.20, AcNRT 1.34, AcNRT 1.41, AcNRT 1.44, AcNRT 1.13, AcNRT 1.30, AcNRT 1.38, AcNRT 2.1, AcNRT1.43, AcNRT 3.1, AcNRT 1.24, AcNRT 2.3, AcNRT1.25, AcNRT 1.23, AcNRT 1.31, AcNRT 1.37, AcNRT 1.8, AcNRT 1.9 and AcNRT 1.12) and nitrogen-deficient MS nutrient solution treatment (AcNRT 1.7, AcNRT 2.1, AcNRT 1.33, AcNRT1.36, AcNRT 1.12, AcNRT 1.2), but only 3 genes were shared by both; 6 days after treatment, 8 genes (AcNRT 1.11, AcNRT 1.19, AcNRT 1.24, AcNRT 2.3, AcNRT 1.25, AcNRT 1.37, AcNRT 1.8 and AcNRT 1.32) were highly expressed in leaves of plants treated with 30 mmol·L^-1NH4NO3 but not in those treated with 60 mmol·L^-1NH4NO3; Except for AcNRT 1.11, AcNRT 1.19 and AcNRT 1.32, these genes were also highly expressed in those with water control. 26th day after treatment, the highly expressed genes were mainly found with 30 mmol·L^-1 NH4NO3 treatment (AcNRT 1.5, AcNRT 1.11, AcNRT 1.19 and AcNRT 1.35), nitrogen- deficient MS treatment (AcNRT 1.14, AcNRT 1.21 and AcNRT 1.22) and water control (AcNRT 1.6 and AcNRT 1.10), but no genes were shared by these three treatments.【Conclusion】The contents of nitrate and the expressions of nitrate transporter genes in various tissues of pineapple plantlet showed diurnal variation patterns, and nitrate application could change the time when the peaks of these two events occurred; the expression of nitrate transporter genes was affected by the nitrogen status of the plant. AcNRT 1.13, AcNRT 2.1 and AcNRT 1.12 genes may be related to leaf nitrogen uptake, and AcNRT 1.14, AcNRT 1.21 and AcNRT 1.22 genes may be related to leaf nitrogen redistribution.