Effects of nitrogen stresses on the nitrogen metabolism and expression of related genes in Poncirus trifoliata and ‘Ziyang Xiangcheng’ (Citrus junos) rootstocks

Abstract：【Objective】‘Ziyang Xiangcheng’[Citrus junos Sieb. ex Tanaka] and trifoliate orange [Poncirus trifoliate (L.) Raf.] are widely used as citrus rootstocks in China. There is little information about the physiological and molecular basis of nitrogen utilization in the two citrus rootstocks. In this paper, the content of inorganic nitrogen, and the enzyme activity and gene expression of nitrogen metabolism were studied in the two rootstocks under nitrogen deficiency and excess stresses in order to reveal the mechanism of citrus nitrogen efficient utilization and lay a foundation for further study on the nitrogen transport function of citrus rootstocks.【Methods】The experiment was conducted in the Citrus Research Institute of the Chinese Academy of Agricultural Sciences, Beibei, Chongqing. The seed coats of‘Ziyang Xiangcheng’and trifoliate orange were removed, and then seeds germinated in relative humidityof 70% at 27 ℃ under darkness for 7 d. Subsequently, the germinated seedlings were transferred to a modified Hoagland solution (pH 6.0±0.5) at 25 ℃ under a 16 h photoperiod (50 µmol·m^-2 ·s ^-1 ) for 30 d. The nutrient solution was replaced every 5 d. Three nitrogen (N) levels including normal nitrogen level (10 mmol·L^-1 , control), N deficiency stress (0 mmol∙L^-1 ) and N excess stress (50 mmol∙L^-1 ) were used in this experiment. The pots were distributed among a randomized complete block design with nine replicates. After 20 d of nitrogen stress treatments, the roots and leaves were collected and immediately frozen in liquid nitrogen, then stored at -80 ℃ for analyzing the Physiological and biochemical parameters related to nitrogen metabolism. The harvested roots and leaves were rinsed with deionized water 3 times, the water was absorbed with quantitative filter paper, then the samples were dried in a forced draft oven at 80 ℃ for 72 h to a constant weight. The dry weight was measured. Then they were stored in a desiccator for analyzing N content. The activities of NR, NiR, NADH-GDH, and GOGAT were determined according to the method of Experimental guide of modern plant physiology, and the expressison level of nitrogen metabolism relevant genes were analyzed by qRT-PCR.【Results】Compared with the control, nitrogen deficiency significantly reduced the nitrogen accumulation in ‘Ziyang Xiangcheng’and excess nitrogen significantly increased the nitrogen accumulation in the two citrus rootstocks. The total nitrogen accumulation of‘Ziyang Xiangcheng’was significantly higher than that of trifoliate orange. This result might be caused by the higher biomass and root hair density of‘Ziyang Xiangcheng’. In the roots and the leaves, the nitrate content of ‘Ziyang Xingcheng’was significantly higher than that of trifoliate orange. The highest nitrate content in the leaves of trifoliate orange and‘Ziyang Xiangcheng’, was found when they were treated with high nitrogen, followed by the control and low nitrogen treatment. Similar results were obtained for the ammonium content. We further measured the expression of nitrogen metabolism-related genes. The relative expression levels of genes such as FdGOGAT, NR, NiR and GDH2 in the leaves of trifoliate orange under N deficiency condition were significantly lower than those of the control, and nitrogen stresses significantly increased the relative expression of genes such as NADH-GOGAT1, NADH-GOGAT2 and NADH-GOGAT3. Meanwhile, the relative expression levels of genes such as NR, NiR, GDH1, GS and GDH2 in‘Ziyang Xiangcheng’leaves under nitrogen stresses were significantly lower than those of the control. the relative expression levels of NR, NADH-GOGAT1, NADH-GOGAT3 and NADP-GDH genes were significantly lower than those of the control, while there was almost no change in expression levels of genes such as Fd- GOGAT and GDH1. Similarly, the relative expression level of the genes Fd- GOGAT, NR, NiR, NADH- GOGAT1, NADH-GOGAT2, NADH-GOGAT3 and NADP-GDH in the roots of‘Ziyang Xiangcheng’under nitrogen stresses were significantly lower than those of the control. The relative expression of the gene GDH2 was not significantly different from that of the control. We also found that the nitrogen deficiency and high nitrogen treatment significantly reduced nitrate reductase, nitrite reductase, glutamyl dehydrogenase and glutamate synthase in the leaves and the roots of‘Ziyang Xiangcheng’and trifoliate orange. The trends in the activities of NR, NiR, NADH-GDH, and GOGAT were consistent with those of NR, NiR, NADH-GDH and GOGAT transcription, respectively. All above results showed that nitrogen stresses significantly reduced the activities of nitrogen-related enzymes in trifoliate orange and‘Ziyang Xiangcheng’, resulting in the reduction of nitrate absorption and ammonium production.【Conclusion】 Nitrogen deficiency significantly reduced the nitrogen content and the activity of nitrogen- related enzymes and the expression of nitrogen- related genes in the roots and the leaves of trifoliate orange and ‘Ziyang Xiangcheng’. Trifoliate oranget showed higher tolerance under high nitrogen treatment compared with‘Ziyang Xiangcheng’, and‘Ziyang Xiangcheng’had higher nitrogen absorption capacitycompared with trifoliate orange.